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Review of literature
The Introduction chapter has been communicated for Invited review as
Saratale R G Saratale G D Parshetti G K Chang J S and Govindwar S P Outlook of bacterial
decolorization and degradation of azo dyes a review International Journal of Environmental
Research and Public Health (2009) (communicated)
11
Review of literature
21 Importance of biological treatment relative to physicochemical methods
Together with industrialization awareness towards the environmental
problems arising due to effluent discharge is of a critical importance A dye
house effluent typically contains 06ndash08 g l-1 dye (Gahr et al 1994) Pollution
caused by dye effluent is mainly due to durability of the dyes in the wastewater
(Jadhav et al 2007) Therefore both color creating and the color using industries
are compelled to search for novel physicochemical treatments and technologies
which are directed particularly towards the decolorization of the dyes from the
effluents There are many reports on the use of physicochemical methods for
color removal from dyes containing effluents (Churchley 1994 Vandevivere et
al 1998 Swaminathan et al 2003 Behnajady et al 2004 Golab et al 2005
Lopez-Grimau and Gutierrez 2005 Santos et al 2007 Wang et al 2007) The
various physicalchemical methods such as adsorption chemical precipitation
photolysis chemical oxidation and reduction electrochemical treatment were
used for the removal of dyes from wastewater effluent (Fig 21)
Fig 21 Treatment methods for the removal of dyes from wastewater effluent
211 Physical and chemical methods
Physical method Chemical method
Adsorption Filtration
Reverse osmosis Electrolysis
Ozonation
Microorganisms
Biological method
Enzymes
Coagulation Floculation
Oxidation
TTrreeaattmmeenntt mmeetthhooddss ffoorr tteexxttiillee eefffflluueennttss
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In advanced countries the coagulationndashflocculation is most widely used
method in textile wastewater treatment plants It can be used either as a
pre-treatment post-treatment or even as a main treatment system (Gaumlhr et al
1994 Marmagne and Coste 1996) This method can efficiently remove mainly
sulphur and disperse dyes whereas acid direct reactive and vat dyes presented
very low coagulationndashflocculation capacity (Marmagne and Coste 1996
Vandevivere et al 1998) Filtration methods such as ultrafiltration
nanofiltration and reverse osmosis have been used for water reuse and chemical
recovery In the textile industry these filtration methods can be used for both
filtering and recycling It is not only pigment-rich streams but also mercerizing
and bleaching wastewaters The specific temperature and chemical composition
of the wastewater determine the type and porosity of the filter to be applied
(Porter 1997) The main drawbacks of membrane technology are the high
investment costs the potential membrane fouling produces secondary waste
streams which need further treatment (Robinson et al 2001) A very good
option would be to consider an anaerobic pre-treatment followed by aerobic and
membrane post-treatments in order to recycle the water
The adsorption methods for the color removal are based on the high
affinity of many dyes for the adsorbent materials found effective for the removal
of a wide range of dyes The main criteria for the selection of an adsorbent
should be based on the characteristics such as high affinity capacity for target
compounds and the possibility of adsorbent regeneration (Karcher et al 2001)
Activated carbon (AC) is the most common adsorbent and found very effective
to the various types of dyes but due to high cost it is not used conventionally
(Walker and Weatherley 1997 Robinson et al 2001) However its requirement
of various types of adsorbents and also their regeneration or disposal makes the
13
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process economically unfeasible (Karcher et al 2001 Anjaneyulu et al 2005)
Some investigators have used alternative materials such as zeolites polymeric
resins ion exchangers and granulated ferric hydroxide In addition to a number
of low-cost adsorbent materials like peat bentonite clay and fly ash have been
investigated for color removal (Ramakrishna and Viraraghavan 1997) Ion
exchange and electro-kinetic coagulation was also found effective but due to
their high sludge producing properties and ineffective to diversity of dyes it
became economically unfeasible hence not accepted widely (Anjaneyulu et al
2005)
Moreover chemical oxidation methods enable destruction or
decomposition of dye molecules In which various types of an oxidizing agent
such as ozone (O3) hydrogen peroxide (H2O2) and permanganate (MnO4) were
used Modification in the chemical composition of a compound or a group of
compounds (for example dyes) takes place in the presence of these oxidizing
agents by which the dye molecule becomes susceptible for the degradation
(Metcalf and Eddy 2003) Ozonation found to be effective due to its high
reactivity with many azo dyes (by breaking azo -N=N- bond) application in
gaseous state no alteration of the reaction volume and providing good color
removal efficiencies (Alaton et al 2002) However it has limitation towards
disperse dyes and those insoluble in water short life time (20 min) low COD
removal as well as the high cost of ozone (Anjaneyulu et al 2005) In advanced
oxidation processes (AOP) (photochemical and photocatalytic) an oxidizing
agents such as O3 and H2O2 or with heterogeneous photocatalysts are used with
catalysts such as TiO2 ZnO2 Mn and Fe in the presence or absence of an
irradiation source are found to be effective by generating (OH-) radical for the
destruction of hazardous dye pollutants (Vandevivere et al 1998 Alaton et al
14
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2002 Al-Kdasi et al 2004 Forgacs et al 2004 Anjaneyulu et al 2005) In
Fenton reaction hydrogen peroxide is added in an acid solution (pH 2ndash3)
containing Fe2+ ions for the generation of free hydroxyl radical (OH-) This
method found relatively cheap and also represents high COD removal and
decolorization efficiencies for both soluble as well as insoluble dyes However
high sludge generation due to the flocculation of reagents and dye molecules still
limit this process (Robinson et al 2001 Van der Zee 2002) The H2O2UV
process is the most effective AOP technology mainly because of high color
removal (up to 95 ) no sludge formation and high COD removal in a short
retention time is achieved (Safarzadeh et al 1997) It is found less effective for
disperse vat dyes and highly colored wastewater Formation of byproducts and
inefficient use of UV light increases the cost of the process (Yang et al 1998)
Electrochemical oxidation found to be very effective in which destruction of
organic compounds resulted into non-hazardous products but high cost of the
electricity limits the process (Robinson et al 2001 Morawski 2002) Thus
majority of color removal techniques work either by concentrating the color into
sludge or by the complete destruction of the colored molecule According to
Integrated Pollution Control (IPC) regulations decolorization systems involving
destruction technologies will persist as the transferal of pollution from one part
of the environment to another need to prevent (Pearce et al 2003) Thus
implementation of physicalchemical methods have inherent drawbacks of
being economically unfeasible (more energy and chemicals) unable to complete
removal of the recalcitrant azo dyes andor their organic metabolites because of
the color fastness stability and resistance of azo dyes to degradation
(Anjaneyulu et al 2005 Dhanve et al 2008) generating a significant amount
of sludge that may cause secondary pollution problems substantially increases
15
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the cost of these treatment methods and involving complicated procedures
(Zhang et al 2004 Forgacs et al 2004 Eichlerovaacute et al 2005 Kalme et al
2007)
212 Biological methods
Bioremediation is the microbial clean up approach is on the front line and
priority research area in the environmental sciences This field has recent origin
and grown exponentially over the last two decades In this system microbes can
acclimatize themselves to toxic wastes and new resistant strains develop
naturally which can transform various toxic chemicals to less harmful forms
The mechanism behind the biodegradation of recalcitrant compounds in the
microbial system is because of the biotransformation enzymes (Saratale et al
2007a) Several reports suggest the degradation of complex organic substances
which can be brought about by an enzymatic mechanism like laccase (Hatvani
and Mecs 2001) lignin peroxidase (Shanmugam et al 1999) NADH-DCIP
reductase (Bhosale et al 2006) tyrosinase (Zhang and Flurkey 1997) hexane
oxidase (Saratale et al 2007b) and aminopyrine N-demethylase (Salokhe and
Govindwar 2003) etc A number of biotechnological approaches have been
suggested by recent research as of potential interest towards combating this
pollution source in an ecoefficient manner mainly the use of bacteria and often
in combination with physicochemical processes Azo dyes constitute the largest
class of dyes used in industries which are xenobiotic in nature and found to be
recalcitrant to biodegradation The isolation of new strains or the adaptation of
existing ones to the decomposition of dyes will probably increase the efficacy of
bioremediation of dyes in the near future The use of microbial or enzymatic
treatment method for the complete decolorization and degradation of an
industrial dyes from textile effluent possess has considerable advantages 1)
16
Review of literature
eco-friendly nature 2) cost-competitive 3) less sludge producing properties 4)
the end products with complete mineralization or non toxic products and 5)
could help to reduce the enormous water consumption compared to
physicochemical methods (Saratale et al 2009b Rai et al 2005) Moreover the
effectiveness of the microbial decolorization depends upon the adaptability and
the activity of selected microorganisms Large number of species has been tested
for the decolorization and mineralization of various dyes and steadily increasing
in recent years (Pandey et al 2007) The isolation of potent species and there by
degradation is one of the interest in biological aspect of effluents treatment
(Mohan et al 2002) A wide variety of microorganisms are capable of
decolorization of a wide range of dyes using wide range of microorganisms
including bacteria (Parshetti et al 2006 Kalyani et al 2008 Telke et al 2008
Dawkar et al 2008 Saratale et al 2009c Jadhav et al 2009) fungi (Fournier
et al 2004 Saratale et al2006 Machado et al 2006 Humnabadkar et al
2008) yeasts (Lucas et al 2006 Jadhav et al 2007 Saratale et al 2009a)
actinomycetes (Parshetti et al 2007) algae (Acuner and Dilek 2004 Yan and
Pan 2004 Parikh and Madamwar 2005 Gupta et al 2006 Daneshvar et al
2007) and plants (Phytoremediation) (Aubert and Schwitzguebel 2004
Ghodake et al 2009 Kagalkar et al 2009) capable of decolorize and even
completely mineralize many azo dyes under certain environmental conditions
213 Fungal decolorization and degradation of textile dyes
Filamentous fungi are found ubiquitous in the environment inhabiting
ecological niches such as soil living plants and organic waste material The
ability of fungi to rapidly adapt their metabolism to varying carbon and nitrogen
sources is an integrated aspect for their survival This metabolic activity
achieved through the production of a large set of intra and extracellular enzymes
17
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able to degrade complex various kinds of organic pollutants (Saratale et al
2007a) In addition to the production and secretion of number of enzymes
filamentous fungi can secrete a great diversity of primary and secondary
metabolites (eg antibiotics) and perform many different complex conversions
such as hydroxylation of complex polyaromatic hydrocarbons organic waste
dye effluents and steroid compounds (McMullan et al 2001 Saratale et al
2007b) Fungal systems appear to be the most appropriate in the treatment of
colored and metallic effluents (Ezeronye and Okerentugba 1999) The ability of
these fungi to degrade such a range of organic compounds results from the
relatively non-specific nature of their ligninolytic enzymes such as lignin
peroxidase (LiP) manganese peroxidase (MnP) and laccase Fungal degradation
of aromatic structures is a secondary metabolic event that starts when nutrients
(C N and S) become limiting (Kirk and Farrell 1987) Therefore while the
enzymes are optimally expressed under starving conditions supplementation of
energy substrates and nutrients are necessary for the propagation of the cultures
(Christian et al 2005)
Most studies on an azo dye biodegradation have focused on the fungal
cultures mainly belonging to white rot fungi and used to develop bioprocesses
for mineralization of azo dyes (Parshetti et al 2007) The Phanerochaete
chrysosporium is the most widely studied of white-rot fungi as well as Trametes
(Coriolus) versicolor Bjerkandera adusta Pleurotus Aspergillus and Phlebia
species and variety of other isolates also studied for the degradation of various
textile dyes (Heinfling et al 1998 Conneely et al 1999 Swamy and Ramsay
1999 Pointing et al 2000 Saratale et al 2006 Humnabadkar et al 2008) A
detailed investigation was also carried out on isolated Geotrichum candidum
Dec1 capable of decolorization a number of anthraquinone dyes (Kim et al
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1995) The broad substrate specificity exhibited by this isolate is due to
production of extracellular peroxidase-type enzymes and glycosylated
haem-based peroxidase (DyP) (Kim and Shoda 1999a)
However application of white-rot fungi for the removal of dyes from
textile wastewater have inherent drawbacks long growth cycle requiring
nitrogen limiting conditions naturally white rot fungi not found in wastewater
hence the enzyme production may be unreliable (Robinson et al 2001) long
hydraulic retention time for complete decolorization still limit the performance
of the fungal decolorization system (Banat et al 1996 Saratale et al 2009b) as
well as preservation in bioreactors will be a matter of concern (Stolz 2001)
214 Decolorization with yeast
Very little work was devoted to exploring the decolorization ability of
yeast mainly studied for the biosorption It was observed that few ascomycetes
yeast species such as Candida tropicalis Debaryomyces polymorphus (Yang et
al 2003) Candida zeylanoides (Martins et al 1999 Ramalho et al 2002) and
Issatchenkia occidentalis (Ramalho et al 2004) perform a putative enzymatic
biodegradation and consequent decolorization of different azo dyes Earlier
Saccharomyces cerevisiae MTCC-463 was reported to involve in the
decolorization of Malachite Green and Methyl Red (Jadhav et al 2006 Jadhav
et al 2007) S cerevisiae cells also showed bioaccumulation of reactive textile
dye (Remazol Blue Remazol Black B and Remazol Red RB) during growth in
molasses (Aksu 2003) Magnetically modified bakers yeast has been used for
the biosorption of Acridine Orange Aniline Blue Crystal Violet Malachite
Green and Safranine acts as a promising dye adsorbent (ˇSafaˇrikova et al
2005) Biological decolorization of triphenylmethane dyes are widely reported
using red yeasts Rhodotorulae sp and Rhodotorulae rubra (Kwasniewska 1995)
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Review of literature
Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane azo
and reactive high exhaust textile dyes as reported earlier (Jadhav et al 2008)
Recently detailed study on the decolorization of Navy Blue HER by using
Trichosporon beigelii NCIM-3326 along with the enzymatic mechanism and
toxicity of degradation products has been reported (Saratale et al 2009a) In a
comparative study on biosorption capacities of different kinds of dried yeasts for
Remazol Blue Candida lipolytica showed the highest dye uptake capacity up to
250 mg g-1 cells (Donmez 2002 Aksu and Donmez 2003)
215 Decolorization with algae
The photosynthetic organisms such as cyanobacteria or algae have a
ubiquitous distribution and observed in all kind of habitats of the world The
literature survey suggests that algae are capable of degrading azo dyes through
an induced form of an azoreductase (Jinqi and Houtian 1992) Color removal by
algae was due to three intrinsically different mechanisms of assimilative
utilization of chromophores for production of algal biomass CO2 and H2O
transformation of colored molecules to non-colored molecules and adsorption of
chromophores on algal biomass Several species of Chlorella (Acuner and Dilek
2004) and Oscillatoria (Jinqi and Houtian 1992) were capable of degrading azo
dyes to their aromatic amines and to further metabolize the aromatic amines to
simpler organic compounds or CO2 Mohan et al (2002) attributes the
decolorization to biosorption followed by bioconversion and biocoagulation It
was reported that more than 30 azo compounds were biodegraded and
decolorized by Chlorella pyrenoidosa Chlorella vulgaris and Oscillateria
tenuis in which azo dyes were decomposed into simpler aromatic amines (Yan
and Pan 2004) Thus the foregoing results could mean that algae can play an
20
Review of literature
important role in the removal of azo dyes and aromatic amines in stabilization
ponds (Banat et al 1996)
216 Decolorization with plant (phytoremediation)
Phytoremediation is considered as a plausible approach for the
remediation of soils and groundwater contaminated with heavy metals and
organic pollutants Recently some studies describe the use of plants for the dye
removal from wastewaters The Rheum rabarbarum mentions a good removal
capacity of sulfonated anthraquinones (Aubert and Schwitzgueacutebel 2004)
Recently narrow-leaved cattails were studied in synthetic reactive dye
wastewater treatment under caustic conditions (Nilratnisakorn et al 2007) Also
Mbuligwe describes a reduction in color of 72-77 in wetlands vegetated with
coco yam plants (Mbuligwe 2005) It was reported that the plant possesses
enzymes that accept anthraquinones as substrates and in cell culture were able to
remove up to 700-800 mg l-1 of anthraquinones with sulphonate groups in
different positions The three plant species (Brassica juncea Sorghum vulgare
and Phaseolus mungo) of different agronomic consequence were evaluated for
the decolorization of the dyes from textile effluent These plants B juncea S
vulgare and P mungo showed textile effluent decolorization up to 79 57 and
53 respectively (Ghodake et al 2008) Similarly an herb Blumea malcommi
was found to degrade textile dye Reactive Red 5B (Kagalkar et al 2009)
However in large scale application of phytoremediation presently faces a
number of obstacles including the level of pollutants tolerated by the plant the
bioavailable fraction of the contaminants and evapotranspiration of volatile
organic pollutants as well as requiring big areas to implant the treatment
(Williams 2002)
217 Bacterial decolorization and degradation of azo dyes
21
Review of literature
Generally the decolorization of azo dyes occurs under conventional
anaerobic anoxic and aerobic conditions by different groups of the bacteria The
mechanism of microbial degradation of azo dyes involves the reductive cleavage
of azo bonds (-N=N-) with the help of azoreductase under anaerobic conditions
resulted into the formation of colorless solutions containing potentially
hazardous-aromatic amines (Chang et al 2000 van der Zee and Villaverde
2005) The resulting intermediate metabolites (eg aromatic amines) are further
degraded aerobically or anaerobically (Seshadri et al 1994) Many recent
researches focus on utilization of microbial biocatalyst to reduce the dye from
the effluent (Hu 1998 Chang et al 2000 Martina Mohorčič 2004) Moreover
the effectiveness of microbial decolorization depends on the adaptability and the
activity of selected microorganisms It has been reported that a wide range of
microorganisms including bacteria fungi yeasts actinomycetes and algae are
capable of degrading azo dyes Moreover most studies on azo dye
biodegradation have focused on the bacteria and fungi The fungal cultures
mainly belonging to white rot fungi have been used to develop bioprocesses for
the mineralization of azo dyes (Parshetti et al 2007) However a long growth
cycle requiring nitrogen limiting conditions and long hydraulic retention time
for complete decolorization still limit the performance of the fungal
decolorization system (Banat et al 1996 Chang et al 2004 Jadhav et al
2008) In contrast bacterial decolorization and degradation of azo dyes has been
of considerable interest since it possesses higher degree of biodegradation and
mineralization diversity towards variety of azo dyes inexpensive and
eco-friendly nature and less sludge producing properties (Verma and Madmawar
2003 Rai et al 2005 Saratale et al 2009c Kalyani et al 2008 Dhanve et al
2008 Saratale et al 2006) Extensive studies have been carried out to determine
22
Review of literature
the role of the diverse groups of bacteria in the decolorization of azo dyes
(Pandey et al 2007)
i) Using pure bacterial culture
The effluents from textile industries are complex containing a wide
variety of dyes and other products such as dispersants acids bases salts
detergents humectants oxidants etc Discharge of these colored effluents into
rivers and lakes results into reduced dissolved oxygen concentration thus
creating anoxic conditions that are lethal to resident organisms Biological
processes provide an alternative to existing technologies because they are more
cost-effective environmentally friendly and do not produce large quantities of
sludge Bacterial decolorization is normally faster compared to fungal system for
the decolorization and mineralization of azo dyes It was observed that the mixed
cultures are apparent as some microbial consortia can collectively carry out
biodegradation tasks that no individual pure strain can undertake successfully
(Nigam et al 1996) However mixed cultures only provide an average
macroscopic view of what is happening in the system and results are not easily
reproduced making thorough effective interpretation difficult For these
reasons a substantial amount of research on the subject of color removal has been
carried out using single bacterial cultures like P mirabilis P luteola
Pseudomonas sp Pseudomonas sp SUK1 and K rosea has shown very
promising results for the dye degradation under anoxic conditions (Chen et al
1999 Chang et al 2001 Yu et al 2001 Kalyani et al 2008 Parshetti et al
2006) In addition there are also several studies describing the decolorization of
reactive dyes mediated by pure bacterial culture such as Exiguobacterium sp
RD3 for Navy Blue HE2R (Reactive Blue 172) (Dhanve et al 2008) Rhizobium
radiobacter MTCC 8161 for Reactive Red 141 (Telke et al 2008) P vulgaris
23
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
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Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
21 Importance of biological treatment relative to physicochemical methods
Together with industrialization awareness towards the environmental
problems arising due to effluent discharge is of a critical importance A dye
house effluent typically contains 06ndash08 g l-1 dye (Gahr et al 1994) Pollution
caused by dye effluent is mainly due to durability of the dyes in the wastewater
(Jadhav et al 2007) Therefore both color creating and the color using industries
are compelled to search for novel physicochemical treatments and technologies
which are directed particularly towards the decolorization of the dyes from the
effluents There are many reports on the use of physicochemical methods for
color removal from dyes containing effluents (Churchley 1994 Vandevivere et
al 1998 Swaminathan et al 2003 Behnajady et al 2004 Golab et al 2005
Lopez-Grimau and Gutierrez 2005 Santos et al 2007 Wang et al 2007) The
various physicalchemical methods such as adsorption chemical precipitation
photolysis chemical oxidation and reduction electrochemical treatment were
used for the removal of dyes from wastewater effluent (Fig 21)
Fig 21 Treatment methods for the removal of dyes from wastewater effluent
211 Physical and chemical methods
Physical method Chemical method
Adsorption Filtration
Reverse osmosis Electrolysis
Ozonation
Microorganisms
Biological method
Enzymes
Coagulation Floculation
Oxidation
TTrreeaattmmeenntt mmeetthhooddss ffoorr tteexxttiillee eefffflluueennttss
12
Review of literature
In advanced countries the coagulationndashflocculation is most widely used
method in textile wastewater treatment plants It can be used either as a
pre-treatment post-treatment or even as a main treatment system (Gaumlhr et al
1994 Marmagne and Coste 1996) This method can efficiently remove mainly
sulphur and disperse dyes whereas acid direct reactive and vat dyes presented
very low coagulationndashflocculation capacity (Marmagne and Coste 1996
Vandevivere et al 1998) Filtration methods such as ultrafiltration
nanofiltration and reverse osmosis have been used for water reuse and chemical
recovery In the textile industry these filtration methods can be used for both
filtering and recycling It is not only pigment-rich streams but also mercerizing
and bleaching wastewaters The specific temperature and chemical composition
of the wastewater determine the type and porosity of the filter to be applied
(Porter 1997) The main drawbacks of membrane technology are the high
investment costs the potential membrane fouling produces secondary waste
streams which need further treatment (Robinson et al 2001) A very good
option would be to consider an anaerobic pre-treatment followed by aerobic and
membrane post-treatments in order to recycle the water
The adsorption methods for the color removal are based on the high
affinity of many dyes for the adsorbent materials found effective for the removal
of a wide range of dyes The main criteria for the selection of an adsorbent
should be based on the characteristics such as high affinity capacity for target
compounds and the possibility of adsorbent regeneration (Karcher et al 2001)
Activated carbon (AC) is the most common adsorbent and found very effective
to the various types of dyes but due to high cost it is not used conventionally
(Walker and Weatherley 1997 Robinson et al 2001) However its requirement
of various types of adsorbents and also their regeneration or disposal makes the
13
Review of literature
process economically unfeasible (Karcher et al 2001 Anjaneyulu et al 2005)
Some investigators have used alternative materials such as zeolites polymeric
resins ion exchangers and granulated ferric hydroxide In addition to a number
of low-cost adsorbent materials like peat bentonite clay and fly ash have been
investigated for color removal (Ramakrishna and Viraraghavan 1997) Ion
exchange and electro-kinetic coagulation was also found effective but due to
their high sludge producing properties and ineffective to diversity of dyes it
became economically unfeasible hence not accepted widely (Anjaneyulu et al
2005)
Moreover chemical oxidation methods enable destruction or
decomposition of dye molecules In which various types of an oxidizing agent
such as ozone (O3) hydrogen peroxide (H2O2) and permanganate (MnO4) were
used Modification in the chemical composition of a compound or a group of
compounds (for example dyes) takes place in the presence of these oxidizing
agents by which the dye molecule becomes susceptible for the degradation
(Metcalf and Eddy 2003) Ozonation found to be effective due to its high
reactivity with many azo dyes (by breaking azo -N=N- bond) application in
gaseous state no alteration of the reaction volume and providing good color
removal efficiencies (Alaton et al 2002) However it has limitation towards
disperse dyes and those insoluble in water short life time (20 min) low COD
removal as well as the high cost of ozone (Anjaneyulu et al 2005) In advanced
oxidation processes (AOP) (photochemical and photocatalytic) an oxidizing
agents such as O3 and H2O2 or with heterogeneous photocatalysts are used with
catalysts such as TiO2 ZnO2 Mn and Fe in the presence or absence of an
irradiation source are found to be effective by generating (OH-) radical for the
destruction of hazardous dye pollutants (Vandevivere et al 1998 Alaton et al
14
Review of literature
2002 Al-Kdasi et al 2004 Forgacs et al 2004 Anjaneyulu et al 2005) In
Fenton reaction hydrogen peroxide is added in an acid solution (pH 2ndash3)
containing Fe2+ ions for the generation of free hydroxyl radical (OH-) This
method found relatively cheap and also represents high COD removal and
decolorization efficiencies for both soluble as well as insoluble dyes However
high sludge generation due to the flocculation of reagents and dye molecules still
limit this process (Robinson et al 2001 Van der Zee 2002) The H2O2UV
process is the most effective AOP technology mainly because of high color
removal (up to 95 ) no sludge formation and high COD removal in a short
retention time is achieved (Safarzadeh et al 1997) It is found less effective for
disperse vat dyes and highly colored wastewater Formation of byproducts and
inefficient use of UV light increases the cost of the process (Yang et al 1998)
Electrochemical oxidation found to be very effective in which destruction of
organic compounds resulted into non-hazardous products but high cost of the
electricity limits the process (Robinson et al 2001 Morawski 2002) Thus
majority of color removal techniques work either by concentrating the color into
sludge or by the complete destruction of the colored molecule According to
Integrated Pollution Control (IPC) regulations decolorization systems involving
destruction technologies will persist as the transferal of pollution from one part
of the environment to another need to prevent (Pearce et al 2003) Thus
implementation of physicalchemical methods have inherent drawbacks of
being economically unfeasible (more energy and chemicals) unable to complete
removal of the recalcitrant azo dyes andor their organic metabolites because of
the color fastness stability and resistance of azo dyes to degradation
(Anjaneyulu et al 2005 Dhanve et al 2008) generating a significant amount
of sludge that may cause secondary pollution problems substantially increases
15
Review of literature
the cost of these treatment methods and involving complicated procedures
(Zhang et al 2004 Forgacs et al 2004 Eichlerovaacute et al 2005 Kalme et al
2007)
212 Biological methods
Bioremediation is the microbial clean up approach is on the front line and
priority research area in the environmental sciences This field has recent origin
and grown exponentially over the last two decades In this system microbes can
acclimatize themselves to toxic wastes and new resistant strains develop
naturally which can transform various toxic chemicals to less harmful forms
The mechanism behind the biodegradation of recalcitrant compounds in the
microbial system is because of the biotransformation enzymes (Saratale et al
2007a) Several reports suggest the degradation of complex organic substances
which can be brought about by an enzymatic mechanism like laccase (Hatvani
and Mecs 2001) lignin peroxidase (Shanmugam et al 1999) NADH-DCIP
reductase (Bhosale et al 2006) tyrosinase (Zhang and Flurkey 1997) hexane
oxidase (Saratale et al 2007b) and aminopyrine N-demethylase (Salokhe and
Govindwar 2003) etc A number of biotechnological approaches have been
suggested by recent research as of potential interest towards combating this
pollution source in an ecoefficient manner mainly the use of bacteria and often
in combination with physicochemical processes Azo dyes constitute the largest
class of dyes used in industries which are xenobiotic in nature and found to be
recalcitrant to biodegradation The isolation of new strains or the adaptation of
existing ones to the decomposition of dyes will probably increase the efficacy of
bioremediation of dyes in the near future The use of microbial or enzymatic
treatment method for the complete decolorization and degradation of an
industrial dyes from textile effluent possess has considerable advantages 1)
16
Review of literature
eco-friendly nature 2) cost-competitive 3) less sludge producing properties 4)
the end products with complete mineralization or non toxic products and 5)
could help to reduce the enormous water consumption compared to
physicochemical methods (Saratale et al 2009b Rai et al 2005) Moreover the
effectiveness of the microbial decolorization depends upon the adaptability and
the activity of selected microorganisms Large number of species has been tested
for the decolorization and mineralization of various dyes and steadily increasing
in recent years (Pandey et al 2007) The isolation of potent species and there by
degradation is one of the interest in biological aspect of effluents treatment
(Mohan et al 2002) A wide variety of microorganisms are capable of
decolorization of a wide range of dyes using wide range of microorganisms
including bacteria (Parshetti et al 2006 Kalyani et al 2008 Telke et al 2008
Dawkar et al 2008 Saratale et al 2009c Jadhav et al 2009) fungi (Fournier
et al 2004 Saratale et al2006 Machado et al 2006 Humnabadkar et al
2008) yeasts (Lucas et al 2006 Jadhav et al 2007 Saratale et al 2009a)
actinomycetes (Parshetti et al 2007) algae (Acuner and Dilek 2004 Yan and
Pan 2004 Parikh and Madamwar 2005 Gupta et al 2006 Daneshvar et al
2007) and plants (Phytoremediation) (Aubert and Schwitzguebel 2004
Ghodake et al 2009 Kagalkar et al 2009) capable of decolorize and even
completely mineralize many azo dyes under certain environmental conditions
213 Fungal decolorization and degradation of textile dyes
Filamentous fungi are found ubiquitous in the environment inhabiting
ecological niches such as soil living plants and organic waste material The
ability of fungi to rapidly adapt their metabolism to varying carbon and nitrogen
sources is an integrated aspect for their survival This metabolic activity
achieved through the production of a large set of intra and extracellular enzymes
17
Review of literature
able to degrade complex various kinds of organic pollutants (Saratale et al
2007a) In addition to the production and secretion of number of enzymes
filamentous fungi can secrete a great diversity of primary and secondary
metabolites (eg antibiotics) and perform many different complex conversions
such as hydroxylation of complex polyaromatic hydrocarbons organic waste
dye effluents and steroid compounds (McMullan et al 2001 Saratale et al
2007b) Fungal systems appear to be the most appropriate in the treatment of
colored and metallic effluents (Ezeronye and Okerentugba 1999) The ability of
these fungi to degrade such a range of organic compounds results from the
relatively non-specific nature of their ligninolytic enzymes such as lignin
peroxidase (LiP) manganese peroxidase (MnP) and laccase Fungal degradation
of aromatic structures is a secondary metabolic event that starts when nutrients
(C N and S) become limiting (Kirk and Farrell 1987) Therefore while the
enzymes are optimally expressed under starving conditions supplementation of
energy substrates and nutrients are necessary for the propagation of the cultures
(Christian et al 2005)
Most studies on an azo dye biodegradation have focused on the fungal
cultures mainly belonging to white rot fungi and used to develop bioprocesses
for mineralization of azo dyes (Parshetti et al 2007) The Phanerochaete
chrysosporium is the most widely studied of white-rot fungi as well as Trametes
(Coriolus) versicolor Bjerkandera adusta Pleurotus Aspergillus and Phlebia
species and variety of other isolates also studied for the degradation of various
textile dyes (Heinfling et al 1998 Conneely et al 1999 Swamy and Ramsay
1999 Pointing et al 2000 Saratale et al 2006 Humnabadkar et al 2008) A
detailed investigation was also carried out on isolated Geotrichum candidum
Dec1 capable of decolorization a number of anthraquinone dyes (Kim et al
18
Review of literature
1995) The broad substrate specificity exhibited by this isolate is due to
production of extracellular peroxidase-type enzymes and glycosylated
haem-based peroxidase (DyP) (Kim and Shoda 1999a)
However application of white-rot fungi for the removal of dyes from
textile wastewater have inherent drawbacks long growth cycle requiring
nitrogen limiting conditions naturally white rot fungi not found in wastewater
hence the enzyme production may be unreliable (Robinson et al 2001) long
hydraulic retention time for complete decolorization still limit the performance
of the fungal decolorization system (Banat et al 1996 Saratale et al 2009b) as
well as preservation in bioreactors will be a matter of concern (Stolz 2001)
214 Decolorization with yeast
Very little work was devoted to exploring the decolorization ability of
yeast mainly studied for the biosorption It was observed that few ascomycetes
yeast species such as Candida tropicalis Debaryomyces polymorphus (Yang et
al 2003) Candida zeylanoides (Martins et al 1999 Ramalho et al 2002) and
Issatchenkia occidentalis (Ramalho et al 2004) perform a putative enzymatic
biodegradation and consequent decolorization of different azo dyes Earlier
Saccharomyces cerevisiae MTCC-463 was reported to involve in the
decolorization of Malachite Green and Methyl Red (Jadhav et al 2006 Jadhav
et al 2007) S cerevisiae cells also showed bioaccumulation of reactive textile
dye (Remazol Blue Remazol Black B and Remazol Red RB) during growth in
molasses (Aksu 2003) Magnetically modified bakers yeast has been used for
the biosorption of Acridine Orange Aniline Blue Crystal Violet Malachite
Green and Safranine acts as a promising dye adsorbent (ˇSafaˇrikova et al
2005) Biological decolorization of triphenylmethane dyes are widely reported
using red yeasts Rhodotorulae sp and Rhodotorulae rubra (Kwasniewska 1995)
19
Review of literature
Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane azo
and reactive high exhaust textile dyes as reported earlier (Jadhav et al 2008)
Recently detailed study on the decolorization of Navy Blue HER by using
Trichosporon beigelii NCIM-3326 along with the enzymatic mechanism and
toxicity of degradation products has been reported (Saratale et al 2009a) In a
comparative study on biosorption capacities of different kinds of dried yeasts for
Remazol Blue Candida lipolytica showed the highest dye uptake capacity up to
250 mg g-1 cells (Donmez 2002 Aksu and Donmez 2003)
215 Decolorization with algae
The photosynthetic organisms such as cyanobacteria or algae have a
ubiquitous distribution and observed in all kind of habitats of the world The
literature survey suggests that algae are capable of degrading azo dyes through
an induced form of an azoreductase (Jinqi and Houtian 1992) Color removal by
algae was due to three intrinsically different mechanisms of assimilative
utilization of chromophores for production of algal biomass CO2 and H2O
transformation of colored molecules to non-colored molecules and adsorption of
chromophores on algal biomass Several species of Chlorella (Acuner and Dilek
2004) and Oscillatoria (Jinqi and Houtian 1992) were capable of degrading azo
dyes to their aromatic amines and to further metabolize the aromatic amines to
simpler organic compounds or CO2 Mohan et al (2002) attributes the
decolorization to biosorption followed by bioconversion and biocoagulation It
was reported that more than 30 azo compounds were biodegraded and
decolorized by Chlorella pyrenoidosa Chlorella vulgaris and Oscillateria
tenuis in which azo dyes were decomposed into simpler aromatic amines (Yan
and Pan 2004) Thus the foregoing results could mean that algae can play an
20
Review of literature
important role in the removal of azo dyes and aromatic amines in stabilization
ponds (Banat et al 1996)
216 Decolorization with plant (phytoremediation)
Phytoremediation is considered as a plausible approach for the
remediation of soils and groundwater contaminated with heavy metals and
organic pollutants Recently some studies describe the use of plants for the dye
removal from wastewaters The Rheum rabarbarum mentions a good removal
capacity of sulfonated anthraquinones (Aubert and Schwitzgueacutebel 2004)
Recently narrow-leaved cattails were studied in synthetic reactive dye
wastewater treatment under caustic conditions (Nilratnisakorn et al 2007) Also
Mbuligwe describes a reduction in color of 72-77 in wetlands vegetated with
coco yam plants (Mbuligwe 2005) It was reported that the plant possesses
enzymes that accept anthraquinones as substrates and in cell culture were able to
remove up to 700-800 mg l-1 of anthraquinones with sulphonate groups in
different positions The three plant species (Brassica juncea Sorghum vulgare
and Phaseolus mungo) of different agronomic consequence were evaluated for
the decolorization of the dyes from textile effluent These plants B juncea S
vulgare and P mungo showed textile effluent decolorization up to 79 57 and
53 respectively (Ghodake et al 2008) Similarly an herb Blumea malcommi
was found to degrade textile dye Reactive Red 5B (Kagalkar et al 2009)
However in large scale application of phytoremediation presently faces a
number of obstacles including the level of pollutants tolerated by the plant the
bioavailable fraction of the contaminants and evapotranspiration of volatile
organic pollutants as well as requiring big areas to implant the treatment
(Williams 2002)
217 Bacterial decolorization and degradation of azo dyes
21
Review of literature
Generally the decolorization of azo dyes occurs under conventional
anaerobic anoxic and aerobic conditions by different groups of the bacteria The
mechanism of microbial degradation of azo dyes involves the reductive cleavage
of azo bonds (-N=N-) with the help of azoreductase under anaerobic conditions
resulted into the formation of colorless solutions containing potentially
hazardous-aromatic amines (Chang et al 2000 van der Zee and Villaverde
2005) The resulting intermediate metabolites (eg aromatic amines) are further
degraded aerobically or anaerobically (Seshadri et al 1994) Many recent
researches focus on utilization of microbial biocatalyst to reduce the dye from
the effluent (Hu 1998 Chang et al 2000 Martina Mohorčič 2004) Moreover
the effectiveness of microbial decolorization depends on the adaptability and the
activity of selected microorganisms It has been reported that a wide range of
microorganisms including bacteria fungi yeasts actinomycetes and algae are
capable of degrading azo dyes Moreover most studies on azo dye
biodegradation have focused on the bacteria and fungi The fungal cultures
mainly belonging to white rot fungi have been used to develop bioprocesses for
the mineralization of azo dyes (Parshetti et al 2007) However a long growth
cycle requiring nitrogen limiting conditions and long hydraulic retention time
for complete decolorization still limit the performance of the fungal
decolorization system (Banat et al 1996 Chang et al 2004 Jadhav et al
2008) In contrast bacterial decolorization and degradation of azo dyes has been
of considerable interest since it possesses higher degree of biodegradation and
mineralization diversity towards variety of azo dyes inexpensive and
eco-friendly nature and less sludge producing properties (Verma and Madmawar
2003 Rai et al 2005 Saratale et al 2009c Kalyani et al 2008 Dhanve et al
2008 Saratale et al 2006) Extensive studies have been carried out to determine
22
Review of literature
the role of the diverse groups of bacteria in the decolorization of azo dyes
(Pandey et al 2007)
i) Using pure bacterial culture
The effluents from textile industries are complex containing a wide
variety of dyes and other products such as dispersants acids bases salts
detergents humectants oxidants etc Discharge of these colored effluents into
rivers and lakes results into reduced dissolved oxygen concentration thus
creating anoxic conditions that are lethal to resident organisms Biological
processes provide an alternative to existing technologies because they are more
cost-effective environmentally friendly and do not produce large quantities of
sludge Bacterial decolorization is normally faster compared to fungal system for
the decolorization and mineralization of azo dyes It was observed that the mixed
cultures are apparent as some microbial consortia can collectively carry out
biodegradation tasks that no individual pure strain can undertake successfully
(Nigam et al 1996) However mixed cultures only provide an average
macroscopic view of what is happening in the system and results are not easily
reproduced making thorough effective interpretation difficult For these
reasons a substantial amount of research on the subject of color removal has been
carried out using single bacterial cultures like P mirabilis P luteola
Pseudomonas sp Pseudomonas sp SUK1 and K rosea has shown very
promising results for the dye degradation under anoxic conditions (Chen et al
1999 Chang et al 2001 Yu et al 2001 Kalyani et al 2008 Parshetti et al
2006) In addition there are also several studies describing the decolorization of
reactive dyes mediated by pure bacterial culture such as Exiguobacterium sp
RD3 for Navy Blue HE2R (Reactive Blue 172) (Dhanve et al 2008) Rhizobium
radiobacter MTCC 8161 for Reactive Red 141 (Telke et al 2008) P vulgaris
23
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
In advanced countries the coagulationndashflocculation is most widely used
method in textile wastewater treatment plants It can be used either as a
pre-treatment post-treatment or even as a main treatment system (Gaumlhr et al
1994 Marmagne and Coste 1996) This method can efficiently remove mainly
sulphur and disperse dyes whereas acid direct reactive and vat dyes presented
very low coagulationndashflocculation capacity (Marmagne and Coste 1996
Vandevivere et al 1998) Filtration methods such as ultrafiltration
nanofiltration and reverse osmosis have been used for water reuse and chemical
recovery In the textile industry these filtration methods can be used for both
filtering and recycling It is not only pigment-rich streams but also mercerizing
and bleaching wastewaters The specific temperature and chemical composition
of the wastewater determine the type and porosity of the filter to be applied
(Porter 1997) The main drawbacks of membrane technology are the high
investment costs the potential membrane fouling produces secondary waste
streams which need further treatment (Robinson et al 2001) A very good
option would be to consider an anaerobic pre-treatment followed by aerobic and
membrane post-treatments in order to recycle the water
The adsorption methods for the color removal are based on the high
affinity of many dyes for the adsorbent materials found effective for the removal
of a wide range of dyes The main criteria for the selection of an adsorbent
should be based on the characteristics such as high affinity capacity for target
compounds and the possibility of adsorbent regeneration (Karcher et al 2001)
Activated carbon (AC) is the most common adsorbent and found very effective
to the various types of dyes but due to high cost it is not used conventionally
(Walker and Weatherley 1997 Robinson et al 2001) However its requirement
of various types of adsorbents and also their regeneration or disposal makes the
13
Review of literature
process economically unfeasible (Karcher et al 2001 Anjaneyulu et al 2005)
Some investigators have used alternative materials such as zeolites polymeric
resins ion exchangers and granulated ferric hydroxide In addition to a number
of low-cost adsorbent materials like peat bentonite clay and fly ash have been
investigated for color removal (Ramakrishna and Viraraghavan 1997) Ion
exchange and electro-kinetic coagulation was also found effective but due to
their high sludge producing properties and ineffective to diversity of dyes it
became economically unfeasible hence not accepted widely (Anjaneyulu et al
2005)
Moreover chemical oxidation methods enable destruction or
decomposition of dye molecules In which various types of an oxidizing agent
such as ozone (O3) hydrogen peroxide (H2O2) and permanganate (MnO4) were
used Modification in the chemical composition of a compound or a group of
compounds (for example dyes) takes place in the presence of these oxidizing
agents by which the dye molecule becomes susceptible for the degradation
(Metcalf and Eddy 2003) Ozonation found to be effective due to its high
reactivity with many azo dyes (by breaking azo -N=N- bond) application in
gaseous state no alteration of the reaction volume and providing good color
removal efficiencies (Alaton et al 2002) However it has limitation towards
disperse dyes and those insoluble in water short life time (20 min) low COD
removal as well as the high cost of ozone (Anjaneyulu et al 2005) In advanced
oxidation processes (AOP) (photochemical and photocatalytic) an oxidizing
agents such as O3 and H2O2 or with heterogeneous photocatalysts are used with
catalysts such as TiO2 ZnO2 Mn and Fe in the presence or absence of an
irradiation source are found to be effective by generating (OH-) radical for the
destruction of hazardous dye pollutants (Vandevivere et al 1998 Alaton et al
14
Review of literature
2002 Al-Kdasi et al 2004 Forgacs et al 2004 Anjaneyulu et al 2005) In
Fenton reaction hydrogen peroxide is added in an acid solution (pH 2ndash3)
containing Fe2+ ions for the generation of free hydroxyl radical (OH-) This
method found relatively cheap and also represents high COD removal and
decolorization efficiencies for both soluble as well as insoluble dyes However
high sludge generation due to the flocculation of reagents and dye molecules still
limit this process (Robinson et al 2001 Van der Zee 2002) The H2O2UV
process is the most effective AOP technology mainly because of high color
removal (up to 95 ) no sludge formation and high COD removal in a short
retention time is achieved (Safarzadeh et al 1997) It is found less effective for
disperse vat dyes and highly colored wastewater Formation of byproducts and
inefficient use of UV light increases the cost of the process (Yang et al 1998)
Electrochemical oxidation found to be very effective in which destruction of
organic compounds resulted into non-hazardous products but high cost of the
electricity limits the process (Robinson et al 2001 Morawski 2002) Thus
majority of color removal techniques work either by concentrating the color into
sludge or by the complete destruction of the colored molecule According to
Integrated Pollution Control (IPC) regulations decolorization systems involving
destruction technologies will persist as the transferal of pollution from one part
of the environment to another need to prevent (Pearce et al 2003) Thus
implementation of physicalchemical methods have inherent drawbacks of
being economically unfeasible (more energy and chemicals) unable to complete
removal of the recalcitrant azo dyes andor their organic metabolites because of
the color fastness stability and resistance of azo dyes to degradation
(Anjaneyulu et al 2005 Dhanve et al 2008) generating a significant amount
of sludge that may cause secondary pollution problems substantially increases
15
Review of literature
the cost of these treatment methods and involving complicated procedures
(Zhang et al 2004 Forgacs et al 2004 Eichlerovaacute et al 2005 Kalme et al
2007)
212 Biological methods
Bioremediation is the microbial clean up approach is on the front line and
priority research area in the environmental sciences This field has recent origin
and grown exponentially over the last two decades In this system microbes can
acclimatize themselves to toxic wastes and new resistant strains develop
naturally which can transform various toxic chemicals to less harmful forms
The mechanism behind the biodegradation of recalcitrant compounds in the
microbial system is because of the biotransformation enzymes (Saratale et al
2007a) Several reports suggest the degradation of complex organic substances
which can be brought about by an enzymatic mechanism like laccase (Hatvani
and Mecs 2001) lignin peroxidase (Shanmugam et al 1999) NADH-DCIP
reductase (Bhosale et al 2006) tyrosinase (Zhang and Flurkey 1997) hexane
oxidase (Saratale et al 2007b) and aminopyrine N-demethylase (Salokhe and
Govindwar 2003) etc A number of biotechnological approaches have been
suggested by recent research as of potential interest towards combating this
pollution source in an ecoefficient manner mainly the use of bacteria and often
in combination with physicochemical processes Azo dyes constitute the largest
class of dyes used in industries which are xenobiotic in nature and found to be
recalcitrant to biodegradation The isolation of new strains or the adaptation of
existing ones to the decomposition of dyes will probably increase the efficacy of
bioremediation of dyes in the near future The use of microbial or enzymatic
treatment method for the complete decolorization and degradation of an
industrial dyes from textile effluent possess has considerable advantages 1)
16
Review of literature
eco-friendly nature 2) cost-competitive 3) less sludge producing properties 4)
the end products with complete mineralization or non toxic products and 5)
could help to reduce the enormous water consumption compared to
physicochemical methods (Saratale et al 2009b Rai et al 2005) Moreover the
effectiveness of the microbial decolorization depends upon the adaptability and
the activity of selected microorganisms Large number of species has been tested
for the decolorization and mineralization of various dyes and steadily increasing
in recent years (Pandey et al 2007) The isolation of potent species and there by
degradation is one of the interest in biological aspect of effluents treatment
(Mohan et al 2002) A wide variety of microorganisms are capable of
decolorization of a wide range of dyes using wide range of microorganisms
including bacteria (Parshetti et al 2006 Kalyani et al 2008 Telke et al 2008
Dawkar et al 2008 Saratale et al 2009c Jadhav et al 2009) fungi (Fournier
et al 2004 Saratale et al2006 Machado et al 2006 Humnabadkar et al
2008) yeasts (Lucas et al 2006 Jadhav et al 2007 Saratale et al 2009a)
actinomycetes (Parshetti et al 2007) algae (Acuner and Dilek 2004 Yan and
Pan 2004 Parikh and Madamwar 2005 Gupta et al 2006 Daneshvar et al
2007) and plants (Phytoremediation) (Aubert and Schwitzguebel 2004
Ghodake et al 2009 Kagalkar et al 2009) capable of decolorize and even
completely mineralize many azo dyes under certain environmental conditions
213 Fungal decolorization and degradation of textile dyes
Filamentous fungi are found ubiquitous in the environment inhabiting
ecological niches such as soil living plants and organic waste material The
ability of fungi to rapidly adapt their metabolism to varying carbon and nitrogen
sources is an integrated aspect for their survival This metabolic activity
achieved through the production of a large set of intra and extracellular enzymes
17
Review of literature
able to degrade complex various kinds of organic pollutants (Saratale et al
2007a) In addition to the production and secretion of number of enzymes
filamentous fungi can secrete a great diversity of primary and secondary
metabolites (eg antibiotics) and perform many different complex conversions
such as hydroxylation of complex polyaromatic hydrocarbons organic waste
dye effluents and steroid compounds (McMullan et al 2001 Saratale et al
2007b) Fungal systems appear to be the most appropriate in the treatment of
colored and metallic effluents (Ezeronye and Okerentugba 1999) The ability of
these fungi to degrade such a range of organic compounds results from the
relatively non-specific nature of their ligninolytic enzymes such as lignin
peroxidase (LiP) manganese peroxidase (MnP) and laccase Fungal degradation
of aromatic structures is a secondary metabolic event that starts when nutrients
(C N and S) become limiting (Kirk and Farrell 1987) Therefore while the
enzymes are optimally expressed under starving conditions supplementation of
energy substrates and nutrients are necessary for the propagation of the cultures
(Christian et al 2005)
Most studies on an azo dye biodegradation have focused on the fungal
cultures mainly belonging to white rot fungi and used to develop bioprocesses
for mineralization of azo dyes (Parshetti et al 2007) The Phanerochaete
chrysosporium is the most widely studied of white-rot fungi as well as Trametes
(Coriolus) versicolor Bjerkandera adusta Pleurotus Aspergillus and Phlebia
species and variety of other isolates also studied for the degradation of various
textile dyes (Heinfling et al 1998 Conneely et al 1999 Swamy and Ramsay
1999 Pointing et al 2000 Saratale et al 2006 Humnabadkar et al 2008) A
detailed investigation was also carried out on isolated Geotrichum candidum
Dec1 capable of decolorization a number of anthraquinone dyes (Kim et al
18
Review of literature
1995) The broad substrate specificity exhibited by this isolate is due to
production of extracellular peroxidase-type enzymes and glycosylated
haem-based peroxidase (DyP) (Kim and Shoda 1999a)
However application of white-rot fungi for the removal of dyes from
textile wastewater have inherent drawbacks long growth cycle requiring
nitrogen limiting conditions naturally white rot fungi not found in wastewater
hence the enzyme production may be unreliable (Robinson et al 2001) long
hydraulic retention time for complete decolorization still limit the performance
of the fungal decolorization system (Banat et al 1996 Saratale et al 2009b) as
well as preservation in bioreactors will be a matter of concern (Stolz 2001)
214 Decolorization with yeast
Very little work was devoted to exploring the decolorization ability of
yeast mainly studied for the biosorption It was observed that few ascomycetes
yeast species such as Candida tropicalis Debaryomyces polymorphus (Yang et
al 2003) Candida zeylanoides (Martins et al 1999 Ramalho et al 2002) and
Issatchenkia occidentalis (Ramalho et al 2004) perform a putative enzymatic
biodegradation and consequent decolorization of different azo dyes Earlier
Saccharomyces cerevisiae MTCC-463 was reported to involve in the
decolorization of Malachite Green and Methyl Red (Jadhav et al 2006 Jadhav
et al 2007) S cerevisiae cells also showed bioaccumulation of reactive textile
dye (Remazol Blue Remazol Black B and Remazol Red RB) during growth in
molasses (Aksu 2003) Magnetically modified bakers yeast has been used for
the biosorption of Acridine Orange Aniline Blue Crystal Violet Malachite
Green and Safranine acts as a promising dye adsorbent (ˇSafaˇrikova et al
2005) Biological decolorization of triphenylmethane dyes are widely reported
using red yeasts Rhodotorulae sp and Rhodotorulae rubra (Kwasniewska 1995)
19
Review of literature
Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane azo
and reactive high exhaust textile dyes as reported earlier (Jadhav et al 2008)
Recently detailed study on the decolorization of Navy Blue HER by using
Trichosporon beigelii NCIM-3326 along with the enzymatic mechanism and
toxicity of degradation products has been reported (Saratale et al 2009a) In a
comparative study on biosorption capacities of different kinds of dried yeasts for
Remazol Blue Candida lipolytica showed the highest dye uptake capacity up to
250 mg g-1 cells (Donmez 2002 Aksu and Donmez 2003)
215 Decolorization with algae
The photosynthetic organisms such as cyanobacteria or algae have a
ubiquitous distribution and observed in all kind of habitats of the world The
literature survey suggests that algae are capable of degrading azo dyes through
an induced form of an azoreductase (Jinqi and Houtian 1992) Color removal by
algae was due to three intrinsically different mechanisms of assimilative
utilization of chromophores for production of algal biomass CO2 and H2O
transformation of colored molecules to non-colored molecules and adsorption of
chromophores on algal biomass Several species of Chlorella (Acuner and Dilek
2004) and Oscillatoria (Jinqi and Houtian 1992) were capable of degrading azo
dyes to their aromatic amines and to further metabolize the aromatic amines to
simpler organic compounds or CO2 Mohan et al (2002) attributes the
decolorization to biosorption followed by bioconversion and biocoagulation It
was reported that more than 30 azo compounds were biodegraded and
decolorized by Chlorella pyrenoidosa Chlorella vulgaris and Oscillateria
tenuis in which azo dyes were decomposed into simpler aromatic amines (Yan
and Pan 2004) Thus the foregoing results could mean that algae can play an
20
Review of literature
important role in the removal of azo dyes and aromatic amines in stabilization
ponds (Banat et al 1996)
216 Decolorization with plant (phytoremediation)
Phytoremediation is considered as a plausible approach for the
remediation of soils and groundwater contaminated with heavy metals and
organic pollutants Recently some studies describe the use of plants for the dye
removal from wastewaters The Rheum rabarbarum mentions a good removal
capacity of sulfonated anthraquinones (Aubert and Schwitzgueacutebel 2004)
Recently narrow-leaved cattails were studied in synthetic reactive dye
wastewater treatment under caustic conditions (Nilratnisakorn et al 2007) Also
Mbuligwe describes a reduction in color of 72-77 in wetlands vegetated with
coco yam plants (Mbuligwe 2005) It was reported that the plant possesses
enzymes that accept anthraquinones as substrates and in cell culture were able to
remove up to 700-800 mg l-1 of anthraquinones with sulphonate groups in
different positions The three plant species (Brassica juncea Sorghum vulgare
and Phaseolus mungo) of different agronomic consequence were evaluated for
the decolorization of the dyes from textile effluent These plants B juncea S
vulgare and P mungo showed textile effluent decolorization up to 79 57 and
53 respectively (Ghodake et al 2008) Similarly an herb Blumea malcommi
was found to degrade textile dye Reactive Red 5B (Kagalkar et al 2009)
However in large scale application of phytoremediation presently faces a
number of obstacles including the level of pollutants tolerated by the plant the
bioavailable fraction of the contaminants and evapotranspiration of volatile
organic pollutants as well as requiring big areas to implant the treatment
(Williams 2002)
217 Bacterial decolorization and degradation of azo dyes
21
Review of literature
Generally the decolorization of azo dyes occurs under conventional
anaerobic anoxic and aerobic conditions by different groups of the bacteria The
mechanism of microbial degradation of azo dyes involves the reductive cleavage
of azo bonds (-N=N-) with the help of azoreductase under anaerobic conditions
resulted into the formation of colorless solutions containing potentially
hazardous-aromatic amines (Chang et al 2000 van der Zee and Villaverde
2005) The resulting intermediate metabolites (eg aromatic amines) are further
degraded aerobically or anaerobically (Seshadri et al 1994) Many recent
researches focus on utilization of microbial biocatalyst to reduce the dye from
the effluent (Hu 1998 Chang et al 2000 Martina Mohorčič 2004) Moreover
the effectiveness of microbial decolorization depends on the adaptability and the
activity of selected microorganisms It has been reported that a wide range of
microorganisms including bacteria fungi yeasts actinomycetes and algae are
capable of degrading azo dyes Moreover most studies on azo dye
biodegradation have focused on the bacteria and fungi The fungal cultures
mainly belonging to white rot fungi have been used to develop bioprocesses for
the mineralization of azo dyes (Parshetti et al 2007) However a long growth
cycle requiring nitrogen limiting conditions and long hydraulic retention time
for complete decolorization still limit the performance of the fungal
decolorization system (Banat et al 1996 Chang et al 2004 Jadhav et al
2008) In contrast bacterial decolorization and degradation of azo dyes has been
of considerable interest since it possesses higher degree of biodegradation and
mineralization diversity towards variety of azo dyes inexpensive and
eco-friendly nature and less sludge producing properties (Verma and Madmawar
2003 Rai et al 2005 Saratale et al 2009c Kalyani et al 2008 Dhanve et al
2008 Saratale et al 2006) Extensive studies have been carried out to determine
22
Review of literature
the role of the diverse groups of bacteria in the decolorization of azo dyes
(Pandey et al 2007)
i) Using pure bacterial culture
The effluents from textile industries are complex containing a wide
variety of dyes and other products such as dispersants acids bases salts
detergents humectants oxidants etc Discharge of these colored effluents into
rivers and lakes results into reduced dissolved oxygen concentration thus
creating anoxic conditions that are lethal to resident organisms Biological
processes provide an alternative to existing technologies because they are more
cost-effective environmentally friendly and do not produce large quantities of
sludge Bacterial decolorization is normally faster compared to fungal system for
the decolorization and mineralization of azo dyes It was observed that the mixed
cultures are apparent as some microbial consortia can collectively carry out
biodegradation tasks that no individual pure strain can undertake successfully
(Nigam et al 1996) However mixed cultures only provide an average
macroscopic view of what is happening in the system and results are not easily
reproduced making thorough effective interpretation difficult For these
reasons a substantial amount of research on the subject of color removal has been
carried out using single bacterial cultures like P mirabilis P luteola
Pseudomonas sp Pseudomonas sp SUK1 and K rosea has shown very
promising results for the dye degradation under anoxic conditions (Chen et al
1999 Chang et al 2001 Yu et al 2001 Kalyani et al 2008 Parshetti et al
2006) In addition there are also several studies describing the decolorization of
reactive dyes mediated by pure bacterial culture such as Exiguobacterium sp
RD3 for Navy Blue HE2R (Reactive Blue 172) (Dhanve et al 2008) Rhizobium
radiobacter MTCC 8161 for Reactive Red 141 (Telke et al 2008) P vulgaris
23
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
process economically unfeasible (Karcher et al 2001 Anjaneyulu et al 2005)
Some investigators have used alternative materials such as zeolites polymeric
resins ion exchangers and granulated ferric hydroxide In addition to a number
of low-cost adsorbent materials like peat bentonite clay and fly ash have been
investigated for color removal (Ramakrishna and Viraraghavan 1997) Ion
exchange and electro-kinetic coagulation was also found effective but due to
their high sludge producing properties and ineffective to diversity of dyes it
became economically unfeasible hence not accepted widely (Anjaneyulu et al
2005)
Moreover chemical oxidation methods enable destruction or
decomposition of dye molecules In which various types of an oxidizing agent
such as ozone (O3) hydrogen peroxide (H2O2) and permanganate (MnO4) were
used Modification in the chemical composition of a compound or a group of
compounds (for example dyes) takes place in the presence of these oxidizing
agents by which the dye molecule becomes susceptible for the degradation
(Metcalf and Eddy 2003) Ozonation found to be effective due to its high
reactivity with many azo dyes (by breaking azo -N=N- bond) application in
gaseous state no alteration of the reaction volume and providing good color
removal efficiencies (Alaton et al 2002) However it has limitation towards
disperse dyes and those insoluble in water short life time (20 min) low COD
removal as well as the high cost of ozone (Anjaneyulu et al 2005) In advanced
oxidation processes (AOP) (photochemical and photocatalytic) an oxidizing
agents such as O3 and H2O2 or with heterogeneous photocatalysts are used with
catalysts such as TiO2 ZnO2 Mn and Fe in the presence or absence of an
irradiation source are found to be effective by generating (OH-) radical for the
destruction of hazardous dye pollutants (Vandevivere et al 1998 Alaton et al
14
Review of literature
2002 Al-Kdasi et al 2004 Forgacs et al 2004 Anjaneyulu et al 2005) In
Fenton reaction hydrogen peroxide is added in an acid solution (pH 2ndash3)
containing Fe2+ ions for the generation of free hydroxyl radical (OH-) This
method found relatively cheap and also represents high COD removal and
decolorization efficiencies for both soluble as well as insoluble dyes However
high sludge generation due to the flocculation of reagents and dye molecules still
limit this process (Robinson et al 2001 Van der Zee 2002) The H2O2UV
process is the most effective AOP technology mainly because of high color
removal (up to 95 ) no sludge formation and high COD removal in a short
retention time is achieved (Safarzadeh et al 1997) It is found less effective for
disperse vat dyes and highly colored wastewater Formation of byproducts and
inefficient use of UV light increases the cost of the process (Yang et al 1998)
Electrochemical oxidation found to be very effective in which destruction of
organic compounds resulted into non-hazardous products but high cost of the
electricity limits the process (Robinson et al 2001 Morawski 2002) Thus
majority of color removal techniques work either by concentrating the color into
sludge or by the complete destruction of the colored molecule According to
Integrated Pollution Control (IPC) regulations decolorization systems involving
destruction technologies will persist as the transferal of pollution from one part
of the environment to another need to prevent (Pearce et al 2003) Thus
implementation of physicalchemical methods have inherent drawbacks of
being economically unfeasible (more energy and chemicals) unable to complete
removal of the recalcitrant azo dyes andor their organic metabolites because of
the color fastness stability and resistance of azo dyes to degradation
(Anjaneyulu et al 2005 Dhanve et al 2008) generating a significant amount
of sludge that may cause secondary pollution problems substantially increases
15
Review of literature
the cost of these treatment methods and involving complicated procedures
(Zhang et al 2004 Forgacs et al 2004 Eichlerovaacute et al 2005 Kalme et al
2007)
212 Biological methods
Bioremediation is the microbial clean up approach is on the front line and
priority research area in the environmental sciences This field has recent origin
and grown exponentially over the last two decades In this system microbes can
acclimatize themselves to toxic wastes and new resistant strains develop
naturally which can transform various toxic chemicals to less harmful forms
The mechanism behind the biodegradation of recalcitrant compounds in the
microbial system is because of the biotransformation enzymes (Saratale et al
2007a) Several reports suggest the degradation of complex organic substances
which can be brought about by an enzymatic mechanism like laccase (Hatvani
and Mecs 2001) lignin peroxidase (Shanmugam et al 1999) NADH-DCIP
reductase (Bhosale et al 2006) tyrosinase (Zhang and Flurkey 1997) hexane
oxidase (Saratale et al 2007b) and aminopyrine N-demethylase (Salokhe and
Govindwar 2003) etc A number of biotechnological approaches have been
suggested by recent research as of potential interest towards combating this
pollution source in an ecoefficient manner mainly the use of bacteria and often
in combination with physicochemical processes Azo dyes constitute the largest
class of dyes used in industries which are xenobiotic in nature and found to be
recalcitrant to biodegradation The isolation of new strains or the adaptation of
existing ones to the decomposition of dyes will probably increase the efficacy of
bioremediation of dyes in the near future The use of microbial or enzymatic
treatment method for the complete decolorization and degradation of an
industrial dyes from textile effluent possess has considerable advantages 1)
16
Review of literature
eco-friendly nature 2) cost-competitive 3) less sludge producing properties 4)
the end products with complete mineralization or non toxic products and 5)
could help to reduce the enormous water consumption compared to
physicochemical methods (Saratale et al 2009b Rai et al 2005) Moreover the
effectiveness of the microbial decolorization depends upon the adaptability and
the activity of selected microorganisms Large number of species has been tested
for the decolorization and mineralization of various dyes and steadily increasing
in recent years (Pandey et al 2007) The isolation of potent species and there by
degradation is one of the interest in biological aspect of effluents treatment
(Mohan et al 2002) A wide variety of microorganisms are capable of
decolorization of a wide range of dyes using wide range of microorganisms
including bacteria (Parshetti et al 2006 Kalyani et al 2008 Telke et al 2008
Dawkar et al 2008 Saratale et al 2009c Jadhav et al 2009) fungi (Fournier
et al 2004 Saratale et al2006 Machado et al 2006 Humnabadkar et al
2008) yeasts (Lucas et al 2006 Jadhav et al 2007 Saratale et al 2009a)
actinomycetes (Parshetti et al 2007) algae (Acuner and Dilek 2004 Yan and
Pan 2004 Parikh and Madamwar 2005 Gupta et al 2006 Daneshvar et al
2007) and plants (Phytoremediation) (Aubert and Schwitzguebel 2004
Ghodake et al 2009 Kagalkar et al 2009) capable of decolorize and even
completely mineralize many azo dyes under certain environmental conditions
213 Fungal decolorization and degradation of textile dyes
Filamentous fungi are found ubiquitous in the environment inhabiting
ecological niches such as soil living plants and organic waste material The
ability of fungi to rapidly adapt their metabolism to varying carbon and nitrogen
sources is an integrated aspect for their survival This metabolic activity
achieved through the production of a large set of intra and extracellular enzymes
17
Review of literature
able to degrade complex various kinds of organic pollutants (Saratale et al
2007a) In addition to the production and secretion of number of enzymes
filamentous fungi can secrete a great diversity of primary and secondary
metabolites (eg antibiotics) and perform many different complex conversions
such as hydroxylation of complex polyaromatic hydrocarbons organic waste
dye effluents and steroid compounds (McMullan et al 2001 Saratale et al
2007b) Fungal systems appear to be the most appropriate in the treatment of
colored and metallic effluents (Ezeronye and Okerentugba 1999) The ability of
these fungi to degrade such a range of organic compounds results from the
relatively non-specific nature of their ligninolytic enzymes such as lignin
peroxidase (LiP) manganese peroxidase (MnP) and laccase Fungal degradation
of aromatic structures is a secondary metabolic event that starts when nutrients
(C N and S) become limiting (Kirk and Farrell 1987) Therefore while the
enzymes are optimally expressed under starving conditions supplementation of
energy substrates and nutrients are necessary for the propagation of the cultures
(Christian et al 2005)
Most studies on an azo dye biodegradation have focused on the fungal
cultures mainly belonging to white rot fungi and used to develop bioprocesses
for mineralization of azo dyes (Parshetti et al 2007) The Phanerochaete
chrysosporium is the most widely studied of white-rot fungi as well as Trametes
(Coriolus) versicolor Bjerkandera adusta Pleurotus Aspergillus and Phlebia
species and variety of other isolates also studied for the degradation of various
textile dyes (Heinfling et al 1998 Conneely et al 1999 Swamy and Ramsay
1999 Pointing et al 2000 Saratale et al 2006 Humnabadkar et al 2008) A
detailed investigation was also carried out on isolated Geotrichum candidum
Dec1 capable of decolorization a number of anthraquinone dyes (Kim et al
18
Review of literature
1995) The broad substrate specificity exhibited by this isolate is due to
production of extracellular peroxidase-type enzymes and glycosylated
haem-based peroxidase (DyP) (Kim and Shoda 1999a)
However application of white-rot fungi for the removal of dyes from
textile wastewater have inherent drawbacks long growth cycle requiring
nitrogen limiting conditions naturally white rot fungi not found in wastewater
hence the enzyme production may be unreliable (Robinson et al 2001) long
hydraulic retention time for complete decolorization still limit the performance
of the fungal decolorization system (Banat et al 1996 Saratale et al 2009b) as
well as preservation in bioreactors will be a matter of concern (Stolz 2001)
214 Decolorization with yeast
Very little work was devoted to exploring the decolorization ability of
yeast mainly studied for the biosorption It was observed that few ascomycetes
yeast species such as Candida tropicalis Debaryomyces polymorphus (Yang et
al 2003) Candida zeylanoides (Martins et al 1999 Ramalho et al 2002) and
Issatchenkia occidentalis (Ramalho et al 2004) perform a putative enzymatic
biodegradation and consequent decolorization of different azo dyes Earlier
Saccharomyces cerevisiae MTCC-463 was reported to involve in the
decolorization of Malachite Green and Methyl Red (Jadhav et al 2006 Jadhav
et al 2007) S cerevisiae cells also showed bioaccumulation of reactive textile
dye (Remazol Blue Remazol Black B and Remazol Red RB) during growth in
molasses (Aksu 2003) Magnetically modified bakers yeast has been used for
the biosorption of Acridine Orange Aniline Blue Crystal Violet Malachite
Green and Safranine acts as a promising dye adsorbent (ˇSafaˇrikova et al
2005) Biological decolorization of triphenylmethane dyes are widely reported
using red yeasts Rhodotorulae sp and Rhodotorulae rubra (Kwasniewska 1995)
19
Review of literature
Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane azo
and reactive high exhaust textile dyes as reported earlier (Jadhav et al 2008)
Recently detailed study on the decolorization of Navy Blue HER by using
Trichosporon beigelii NCIM-3326 along with the enzymatic mechanism and
toxicity of degradation products has been reported (Saratale et al 2009a) In a
comparative study on biosorption capacities of different kinds of dried yeasts for
Remazol Blue Candida lipolytica showed the highest dye uptake capacity up to
250 mg g-1 cells (Donmez 2002 Aksu and Donmez 2003)
215 Decolorization with algae
The photosynthetic organisms such as cyanobacteria or algae have a
ubiquitous distribution and observed in all kind of habitats of the world The
literature survey suggests that algae are capable of degrading azo dyes through
an induced form of an azoreductase (Jinqi and Houtian 1992) Color removal by
algae was due to three intrinsically different mechanisms of assimilative
utilization of chromophores for production of algal biomass CO2 and H2O
transformation of colored molecules to non-colored molecules and adsorption of
chromophores on algal biomass Several species of Chlorella (Acuner and Dilek
2004) and Oscillatoria (Jinqi and Houtian 1992) were capable of degrading azo
dyes to their aromatic amines and to further metabolize the aromatic amines to
simpler organic compounds or CO2 Mohan et al (2002) attributes the
decolorization to biosorption followed by bioconversion and biocoagulation It
was reported that more than 30 azo compounds were biodegraded and
decolorized by Chlorella pyrenoidosa Chlorella vulgaris and Oscillateria
tenuis in which azo dyes were decomposed into simpler aromatic amines (Yan
and Pan 2004) Thus the foregoing results could mean that algae can play an
20
Review of literature
important role in the removal of azo dyes and aromatic amines in stabilization
ponds (Banat et al 1996)
216 Decolorization with plant (phytoremediation)
Phytoremediation is considered as a plausible approach for the
remediation of soils and groundwater contaminated with heavy metals and
organic pollutants Recently some studies describe the use of plants for the dye
removal from wastewaters The Rheum rabarbarum mentions a good removal
capacity of sulfonated anthraquinones (Aubert and Schwitzgueacutebel 2004)
Recently narrow-leaved cattails were studied in synthetic reactive dye
wastewater treatment under caustic conditions (Nilratnisakorn et al 2007) Also
Mbuligwe describes a reduction in color of 72-77 in wetlands vegetated with
coco yam plants (Mbuligwe 2005) It was reported that the plant possesses
enzymes that accept anthraquinones as substrates and in cell culture were able to
remove up to 700-800 mg l-1 of anthraquinones with sulphonate groups in
different positions The three plant species (Brassica juncea Sorghum vulgare
and Phaseolus mungo) of different agronomic consequence were evaluated for
the decolorization of the dyes from textile effluent These plants B juncea S
vulgare and P mungo showed textile effluent decolorization up to 79 57 and
53 respectively (Ghodake et al 2008) Similarly an herb Blumea malcommi
was found to degrade textile dye Reactive Red 5B (Kagalkar et al 2009)
However in large scale application of phytoremediation presently faces a
number of obstacles including the level of pollutants tolerated by the plant the
bioavailable fraction of the contaminants and evapotranspiration of volatile
organic pollutants as well as requiring big areas to implant the treatment
(Williams 2002)
217 Bacterial decolorization and degradation of azo dyes
21
Review of literature
Generally the decolorization of azo dyes occurs under conventional
anaerobic anoxic and aerobic conditions by different groups of the bacteria The
mechanism of microbial degradation of azo dyes involves the reductive cleavage
of azo bonds (-N=N-) with the help of azoreductase under anaerobic conditions
resulted into the formation of colorless solutions containing potentially
hazardous-aromatic amines (Chang et al 2000 van der Zee and Villaverde
2005) The resulting intermediate metabolites (eg aromatic amines) are further
degraded aerobically or anaerobically (Seshadri et al 1994) Many recent
researches focus on utilization of microbial biocatalyst to reduce the dye from
the effluent (Hu 1998 Chang et al 2000 Martina Mohorčič 2004) Moreover
the effectiveness of microbial decolorization depends on the adaptability and the
activity of selected microorganisms It has been reported that a wide range of
microorganisms including bacteria fungi yeasts actinomycetes and algae are
capable of degrading azo dyes Moreover most studies on azo dye
biodegradation have focused on the bacteria and fungi The fungal cultures
mainly belonging to white rot fungi have been used to develop bioprocesses for
the mineralization of azo dyes (Parshetti et al 2007) However a long growth
cycle requiring nitrogen limiting conditions and long hydraulic retention time
for complete decolorization still limit the performance of the fungal
decolorization system (Banat et al 1996 Chang et al 2004 Jadhav et al
2008) In contrast bacterial decolorization and degradation of azo dyes has been
of considerable interest since it possesses higher degree of biodegradation and
mineralization diversity towards variety of azo dyes inexpensive and
eco-friendly nature and less sludge producing properties (Verma and Madmawar
2003 Rai et al 2005 Saratale et al 2009c Kalyani et al 2008 Dhanve et al
2008 Saratale et al 2006) Extensive studies have been carried out to determine
22
Review of literature
the role of the diverse groups of bacteria in the decolorization of azo dyes
(Pandey et al 2007)
i) Using pure bacterial culture
The effluents from textile industries are complex containing a wide
variety of dyes and other products such as dispersants acids bases salts
detergents humectants oxidants etc Discharge of these colored effluents into
rivers and lakes results into reduced dissolved oxygen concentration thus
creating anoxic conditions that are lethal to resident organisms Biological
processes provide an alternative to existing technologies because they are more
cost-effective environmentally friendly and do not produce large quantities of
sludge Bacterial decolorization is normally faster compared to fungal system for
the decolorization and mineralization of azo dyes It was observed that the mixed
cultures are apparent as some microbial consortia can collectively carry out
biodegradation tasks that no individual pure strain can undertake successfully
(Nigam et al 1996) However mixed cultures only provide an average
macroscopic view of what is happening in the system and results are not easily
reproduced making thorough effective interpretation difficult For these
reasons a substantial amount of research on the subject of color removal has been
carried out using single bacterial cultures like P mirabilis P luteola
Pseudomonas sp Pseudomonas sp SUK1 and K rosea has shown very
promising results for the dye degradation under anoxic conditions (Chen et al
1999 Chang et al 2001 Yu et al 2001 Kalyani et al 2008 Parshetti et al
2006) In addition there are also several studies describing the decolorization of
reactive dyes mediated by pure bacterial culture such as Exiguobacterium sp
RD3 for Navy Blue HE2R (Reactive Blue 172) (Dhanve et al 2008) Rhizobium
radiobacter MTCC 8161 for Reactive Red 141 (Telke et al 2008) P vulgaris
23
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
2002 Al-Kdasi et al 2004 Forgacs et al 2004 Anjaneyulu et al 2005) In
Fenton reaction hydrogen peroxide is added in an acid solution (pH 2ndash3)
containing Fe2+ ions for the generation of free hydroxyl radical (OH-) This
method found relatively cheap and also represents high COD removal and
decolorization efficiencies for both soluble as well as insoluble dyes However
high sludge generation due to the flocculation of reagents and dye molecules still
limit this process (Robinson et al 2001 Van der Zee 2002) The H2O2UV
process is the most effective AOP technology mainly because of high color
removal (up to 95 ) no sludge formation and high COD removal in a short
retention time is achieved (Safarzadeh et al 1997) It is found less effective for
disperse vat dyes and highly colored wastewater Formation of byproducts and
inefficient use of UV light increases the cost of the process (Yang et al 1998)
Electrochemical oxidation found to be very effective in which destruction of
organic compounds resulted into non-hazardous products but high cost of the
electricity limits the process (Robinson et al 2001 Morawski 2002) Thus
majority of color removal techniques work either by concentrating the color into
sludge or by the complete destruction of the colored molecule According to
Integrated Pollution Control (IPC) regulations decolorization systems involving
destruction technologies will persist as the transferal of pollution from one part
of the environment to another need to prevent (Pearce et al 2003) Thus
implementation of physicalchemical methods have inherent drawbacks of
being economically unfeasible (more energy and chemicals) unable to complete
removal of the recalcitrant azo dyes andor their organic metabolites because of
the color fastness stability and resistance of azo dyes to degradation
(Anjaneyulu et al 2005 Dhanve et al 2008) generating a significant amount
of sludge that may cause secondary pollution problems substantially increases
15
Review of literature
the cost of these treatment methods and involving complicated procedures
(Zhang et al 2004 Forgacs et al 2004 Eichlerovaacute et al 2005 Kalme et al
2007)
212 Biological methods
Bioremediation is the microbial clean up approach is on the front line and
priority research area in the environmental sciences This field has recent origin
and grown exponentially over the last two decades In this system microbes can
acclimatize themselves to toxic wastes and new resistant strains develop
naturally which can transform various toxic chemicals to less harmful forms
The mechanism behind the biodegradation of recalcitrant compounds in the
microbial system is because of the biotransformation enzymes (Saratale et al
2007a) Several reports suggest the degradation of complex organic substances
which can be brought about by an enzymatic mechanism like laccase (Hatvani
and Mecs 2001) lignin peroxidase (Shanmugam et al 1999) NADH-DCIP
reductase (Bhosale et al 2006) tyrosinase (Zhang and Flurkey 1997) hexane
oxidase (Saratale et al 2007b) and aminopyrine N-demethylase (Salokhe and
Govindwar 2003) etc A number of biotechnological approaches have been
suggested by recent research as of potential interest towards combating this
pollution source in an ecoefficient manner mainly the use of bacteria and often
in combination with physicochemical processes Azo dyes constitute the largest
class of dyes used in industries which are xenobiotic in nature and found to be
recalcitrant to biodegradation The isolation of new strains or the adaptation of
existing ones to the decomposition of dyes will probably increase the efficacy of
bioremediation of dyes in the near future The use of microbial or enzymatic
treatment method for the complete decolorization and degradation of an
industrial dyes from textile effluent possess has considerable advantages 1)
16
Review of literature
eco-friendly nature 2) cost-competitive 3) less sludge producing properties 4)
the end products with complete mineralization or non toxic products and 5)
could help to reduce the enormous water consumption compared to
physicochemical methods (Saratale et al 2009b Rai et al 2005) Moreover the
effectiveness of the microbial decolorization depends upon the adaptability and
the activity of selected microorganisms Large number of species has been tested
for the decolorization and mineralization of various dyes and steadily increasing
in recent years (Pandey et al 2007) The isolation of potent species and there by
degradation is one of the interest in biological aspect of effluents treatment
(Mohan et al 2002) A wide variety of microorganisms are capable of
decolorization of a wide range of dyes using wide range of microorganisms
including bacteria (Parshetti et al 2006 Kalyani et al 2008 Telke et al 2008
Dawkar et al 2008 Saratale et al 2009c Jadhav et al 2009) fungi (Fournier
et al 2004 Saratale et al2006 Machado et al 2006 Humnabadkar et al
2008) yeasts (Lucas et al 2006 Jadhav et al 2007 Saratale et al 2009a)
actinomycetes (Parshetti et al 2007) algae (Acuner and Dilek 2004 Yan and
Pan 2004 Parikh and Madamwar 2005 Gupta et al 2006 Daneshvar et al
2007) and plants (Phytoremediation) (Aubert and Schwitzguebel 2004
Ghodake et al 2009 Kagalkar et al 2009) capable of decolorize and even
completely mineralize many azo dyes under certain environmental conditions
213 Fungal decolorization and degradation of textile dyes
Filamentous fungi are found ubiquitous in the environment inhabiting
ecological niches such as soil living plants and organic waste material The
ability of fungi to rapidly adapt their metabolism to varying carbon and nitrogen
sources is an integrated aspect for their survival This metabolic activity
achieved through the production of a large set of intra and extracellular enzymes
17
Review of literature
able to degrade complex various kinds of organic pollutants (Saratale et al
2007a) In addition to the production and secretion of number of enzymes
filamentous fungi can secrete a great diversity of primary and secondary
metabolites (eg antibiotics) and perform many different complex conversions
such as hydroxylation of complex polyaromatic hydrocarbons organic waste
dye effluents and steroid compounds (McMullan et al 2001 Saratale et al
2007b) Fungal systems appear to be the most appropriate in the treatment of
colored and metallic effluents (Ezeronye and Okerentugba 1999) The ability of
these fungi to degrade such a range of organic compounds results from the
relatively non-specific nature of their ligninolytic enzymes such as lignin
peroxidase (LiP) manganese peroxidase (MnP) and laccase Fungal degradation
of aromatic structures is a secondary metabolic event that starts when nutrients
(C N and S) become limiting (Kirk and Farrell 1987) Therefore while the
enzymes are optimally expressed under starving conditions supplementation of
energy substrates and nutrients are necessary for the propagation of the cultures
(Christian et al 2005)
Most studies on an azo dye biodegradation have focused on the fungal
cultures mainly belonging to white rot fungi and used to develop bioprocesses
for mineralization of azo dyes (Parshetti et al 2007) The Phanerochaete
chrysosporium is the most widely studied of white-rot fungi as well as Trametes
(Coriolus) versicolor Bjerkandera adusta Pleurotus Aspergillus and Phlebia
species and variety of other isolates also studied for the degradation of various
textile dyes (Heinfling et al 1998 Conneely et al 1999 Swamy and Ramsay
1999 Pointing et al 2000 Saratale et al 2006 Humnabadkar et al 2008) A
detailed investigation was also carried out on isolated Geotrichum candidum
Dec1 capable of decolorization a number of anthraquinone dyes (Kim et al
18
Review of literature
1995) The broad substrate specificity exhibited by this isolate is due to
production of extracellular peroxidase-type enzymes and glycosylated
haem-based peroxidase (DyP) (Kim and Shoda 1999a)
However application of white-rot fungi for the removal of dyes from
textile wastewater have inherent drawbacks long growth cycle requiring
nitrogen limiting conditions naturally white rot fungi not found in wastewater
hence the enzyme production may be unreliable (Robinson et al 2001) long
hydraulic retention time for complete decolorization still limit the performance
of the fungal decolorization system (Banat et al 1996 Saratale et al 2009b) as
well as preservation in bioreactors will be a matter of concern (Stolz 2001)
214 Decolorization with yeast
Very little work was devoted to exploring the decolorization ability of
yeast mainly studied for the biosorption It was observed that few ascomycetes
yeast species such as Candida tropicalis Debaryomyces polymorphus (Yang et
al 2003) Candida zeylanoides (Martins et al 1999 Ramalho et al 2002) and
Issatchenkia occidentalis (Ramalho et al 2004) perform a putative enzymatic
biodegradation and consequent decolorization of different azo dyes Earlier
Saccharomyces cerevisiae MTCC-463 was reported to involve in the
decolorization of Malachite Green and Methyl Red (Jadhav et al 2006 Jadhav
et al 2007) S cerevisiae cells also showed bioaccumulation of reactive textile
dye (Remazol Blue Remazol Black B and Remazol Red RB) during growth in
molasses (Aksu 2003) Magnetically modified bakers yeast has been used for
the biosorption of Acridine Orange Aniline Blue Crystal Violet Malachite
Green and Safranine acts as a promising dye adsorbent (ˇSafaˇrikova et al
2005) Biological decolorization of triphenylmethane dyes are widely reported
using red yeasts Rhodotorulae sp and Rhodotorulae rubra (Kwasniewska 1995)
19
Review of literature
Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane azo
and reactive high exhaust textile dyes as reported earlier (Jadhav et al 2008)
Recently detailed study on the decolorization of Navy Blue HER by using
Trichosporon beigelii NCIM-3326 along with the enzymatic mechanism and
toxicity of degradation products has been reported (Saratale et al 2009a) In a
comparative study on biosorption capacities of different kinds of dried yeasts for
Remazol Blue Candida lipolytica showed the highest dye uptake capacity up to
250 mg g-1 cells (Donmez 2002 Aksu and Donmez 2003)
215 Decolorization with algae
The photosynthetic organisms such as cyanobacteria or algae have a
ubiquitous distribution and observed in all kind of habitats of the world The
literature survey suggests that algae are capable of degrading azo dyes through
an induced form of an azoreductase (Jinqi and Houtian 1992) Color removal by
algae was due to three intrinsically different mechanisms of assimilative
utilization of chromophores for production of algal biomass CO2 and H2O
transformation of colored molecules to non-colored molecules and adsorption of
chromophores on algal biomass Several species of Chlorella (Acuner and Dilek
2004) and Oscillatoria (Jinqi and Houtian 1992) were capable of degrading azo
dyes to their aromatic amines and to further metabolize the aromatic amines to
simpler organic compounds or CO2 Mohan et al (2002) attributes the
decolorization to biosorption followed by bioconversion and biocoagulation It
was reported that more than 30 azo compounds were biodegraded and
decolorized by Chlorella pyrenoidosa Chlorella vulgaris and Oscillateria
tenuis in which azo dyes were decomposed into simpler aromatic amines (Yan
and Pan 2004) Thus the foregoing results could mean that algae can play an
20
Review of literature
important role in the removal of azo dyes and aromatic amines in stabilization
ponds (Banat et al 1996)
216 Decolorization with plant (phytoremediation)
Phytoremediation is considered as a plausible approach for the
remediation of soils and groundwater contaminated with heavy metals and
organic pollutants Recently some studies describe the use of plants for the dye
removal from wastewaters The Rheum rabarbarum mentions a good removal
capacity of sulfonated anthraquinones (Aubert and Schwitzgueacutebel 2004)
Recently narrow-leaved cattails were studied in synthetic reactive dye
wastewater treatment under caustic conditions (Nilratnisakorn et al 2007) Also
Mbuligwe describes a reduction in color of 72-77 in wetlands vegetated with
coco yam plants (Mbuligwe 2005) It was reported that the plant possesses
enzymes that accept anthraquinones as substrates and in cell culture were able to
remove up to 700-800 mg l-1 of anthraquinones with sulphonate groups in
different positions The three plant species (Brassica juncea Sorghum vulgare
and Phaseolus mungo) of different agronomic consequence were evaluated for
the decolorization of the dyes from textile effluent These plants B juncea S
vulgare and P mungo showed textile effluent decolorization up to 79 57 and
53 respectively (Ghodake et al 2008) Similarly an herb Blumea malcommi
was found to degrade textile dye Reactive Red 5B (Kagalkar et al 2009)
However in large scale application of phytoremediation presently faces a
number of obstacles including the level of pollutants tolerated by the plant the
bioavailable fraction of the contaminants and evapotranspiration of volatile
organic pollutants as well as requiring big areas to implant the treatment
(Williams 2002)
217 Bacterial decolorization and degradation of azo dyes
21
Review of literature
Generally the decolorization of azo dyes occurs under conventional
anaerobic anoxic and aerobic conditions by different groups of the bacteria The
mechanism of microbial degradation of azo dyes involves the reductive cleavage
of azo bonds (-N=N-) with the help of azoreductase under anaerobic conditions
resulted into the formation of colorless solutions containing potentially
hazardous-aromatic amines (Chang et al 2000 van der Zee and Villaverde
2005) The resulting intermediate metabolites (eg aromatic amines) are further
degraded aerobically or anaerobically (Seshadri et al 1994) Many recent
researches focus on utilization of microbial biocatalyst to reduce the dye from
the effluent (Hu 1998 Chang et al 2000 Martina Mohorčič 2004) Moreover
the effectiveness of microbial decolorization depends on the adaptability and the
activity of selected microorganisms It has been reported that a wide range of
microorganisms including bacteria fungi yeasts actinomycetes and algae are
capable of degrading azo dyes Moreover most studies on azo dye
biodegradation have focused on the bacteria and fungi The fungal cultures
mainly belonging to white rot fungi have been used to develop bioprocesses for
the mineralization of azo dyes (Parshetti et al 2007) However a long growth
cycle requiring nitrogen limiting conditions and long hydraulic retention time
for complete decolorization still limit the performance of the fungal
decolorization system (Banat et al 1996 Chang et al 2004 Jadhav et al
2008) In contrast bacterial decolorization and degradation of azo dyes has been
of considerable interest since it possesses higher degree of biodegradation and
mineralization diversity towards variety of azo dyes inexpensive and
eco-friendly nature and less sludge producing properties (Verma and Madmawar
2003 Rai et al 2005 Saratale et al 2009c Kalyani et al 2008 Dhanve et al
2008 Saratale et al 2006) Extensive studies have been carried out to determine
22
Review of literature
the role of the diverse groups of bacteria in the decolorization of azo dyes
(Pandey et al 2007)
i) Using pure bacterial culture
The effluents from textile industries are complex containing a wide
variety of dyes and other products such as dispersants acids bases salts
detergents humectants oxidants etc Discharge of these colored effluents into
rivers and lakes results into reduced dissolved oxygen concentration thus
creating anoxic conditions that are lethal to resident organisms Biological
processes provide an alternative to existing technologies because they are more
cost-effective environmentally friendly and do not produce large quantities of
sludge Bacterial decolorization is normally faster compared to fungal system for
the decolorization and mineralization of azo dyes It was observed that the mixed
cultures are apparent as some microbial consortia can collectively carry out
biodegradation tasks that no individual pure strain can undertake successfully
(Nigam et al 1996) However mixed cultures only provide an average
macroscopic view of what is happening in the system and results are not easily
reproduced making thorough effective interpretation difficult For these
reasons a substantial amount of research on the subject of color removal has been
carried out using single bacterial cultures like P mirabilis P luteola
Pseudomonas sp Pseudomonas sp SUK1 and K rosea has shown very
promising results for the dye degradation under anoxic conditions (Chen et al
1999 Chang et al 2001 Yu et al 2001 Kalyani et al 2008 Parshetti et al
2006) In addition there are also several studies describing the decolorization of
reactive dyes mediated by pure bacterial culture such as Exiguobacterium sp
RD3 for Navy Blue HE2R (Reactive Blue 172) (Dhanve et al 2008) Rhizobium
radiobacter MTCC 8161 for Reactive Red 141 (Telke et al 2008) P vulgaris
23
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
the cost of these treatment methods and involving complicated procedures
(Zhang et al 2004 Forgacs et al 2004 Eichlerovaacute et al 2005 Kalme et al
2007)
212 Biological methods
Bioremediation is the microbial clean up approach is on the front line and
priority research area in the environmental sciences This field has recent origin
and grown exponentially over the last two decades In this system microbes can
acclimatize themselves to toxic wastes and new resistant strains develop
naturally which can transform various toxic chemicals to less harmful forms
The mechanism behind the biodegradation of recalcitrant compounds in the
microbial system is because of the biotransformation enzymes (Saratale et al
2007a) Several reports suggest the degradation of complex organic substances
which can be brought about by an enzymatic mechanism like laccase (Hatvani
and Mecs 2001) lignin peroxidase (Shanmugam et al 1999) NADH-DCIP
reductase (Bhosale et al 2006) tyrosinase (Zhang and Flurkey 1997) hexane
oxidase (Saratale et al 2007b) and aminopyrine N-demethylase (Salokhe and
Govindwar 2003) etc A number of biotechnological approaches have been
suggested by recent research as of potential interest towards combating this
pollution source in an ecoefficient manner mainly the use of bacteria and often
in combination with physicochemical processes Azo dyes constitute the largest
class of dyes used in industries which are xenobiotic in nature and found to be
recalcitrant to biodegradation The isolation of new strains or the adaptation of
existing ones to the decomposition of dyes will probably increase the efficacy of
bioremediation of dyes in the near future The use of microbial or enzymatic
treatment method for the complete decolorization and degradation of an
industrial dyes from textile effluent possess has considerable advantages 1)
16
Review of literature
eco-friendly nature 2) cost-competitive 3) less sludge producing properties 4)
the end products with complete mineralization or non toxic products and 5)
could help to reduce the enormous water consumption compared to
physicochemical methods (Saratale et al 2009b Rai et al 2005) Moreover the
effectiveness of the microbial decolorization depends upon the adaptability and
the activity of selected microorganisms Large number of species has been tested
for the decolorization and mineralization of various dyes and steadily increasing
in recent years (Pandey et al 2007) The isolation of potent species and there by
degradation is one of the interest in biological aspect of effluents treatment
(Mohan et al 2002) A wide variety of microorganisms are capable of
decolorization of a wide range of dyes using wide range of microorganisms
including bacteria (Parshetti et al 2006 Kalyani et al 2008 Telke et al 2008
Dawkar et al 2008 Saratale et al 2009c Jadhav et al 2009) fungi (Fournier
et al 2004 Saratale et al2006 Machado et al 2006 Humnabadkar et al
2008) yeasts (Lucas et al 2006 Jadhav et al 2007 Saratale et al 2009a)
actinomycetes (Parshetti et al 2007) algae (Acuner and Dilek 2004 Yan and
Pan 2004 Parikh and Madamwar 2005 Gupta et al 2006 Daneshvar et al
2007) and plants (Phytoremediation) (Aubert and Schwitzguebel 2004
Ghodake et al 2009 Kagalkar et al 2009) capable of decolorize and even
completely mineralize many azo dyes under certain environmental conditions
213 Fungal decolorization and degradation of textile dyes
Filamentous fungi are found ubiquitous in the environment inhabiting
ecological niches such as soil living plants and organic waste material The
ability of fungi to rapidly adapt their metabolism to varying carbon and nitrogen
sources is an integrated aspect for their survival This metabolic activity
achieved through the production of a large set of intra and extracellular enzymes
17
Review of literature
able to degrade complex various kinds of organic pollutants (Saratale et al
2007a) In addition to the production and secretion of number of enzymes
filamentous fungi can secrete a great diversity of primary and secondary
metabolites (eg antibiotics) and perform many different complex conversions
such as hydroxylation of complex polyaromatic hydrocarbons organic waste
dye effluents and steroid compounds (McMullan et al 2001 Saratale et al
2007b) Fungal systems appear to be the most appropriate in the treatment of
colored and metallic effluents (Ezeronye and Okerentugba 1999) The ability of
these fungi to degrade such a range of organic compounds results from the
relatively non-specific nature of their ligninolytic enzymes such as lignin
peroxidase (LiP) manganese peroxidase (MnP) and laccase Fungal degradation
of aromatic structures is a secondary metabolic event that starts when nutrients
(C N and S) become limiting (Kirk and Farrell 1987) Therefore while the
enzymes are optimally expressed under starving conditions supplementation of
energy substrates and nutrients are necessary for the propagation of the cultures
(Christian et al 2005)
Most studies on an azo dye biodegradation have focused on the fungal
cultures mainly belonging to white rot fungi and used to develop bioprocesses
for mineralization of azo dyes (Parshetti et al 2007) The Phanerochaete
chrysosporium is the most widely studied of white-rot fungi as well as Trametes
(Coriolus) versicolor Bjerkandera adusta Pleurotus Aspergillus and Phlebia
species and variety of other isolates also studied for the degradation of various
textile dyes (Heinfling et al 1998 Conneely et al 1999 Swamy and Ramsay
1999 Pointing et al 2000 Saratale et al 2006 Humnabadkar et al 2008) A
detailed investigation was also carried out on isolated Geotrichum candidum
Dec1 capable of decolorization a number of anthraquinone dyes (Kim et al
18
Review of literature
1995) The broad substrate specificity exhibited by this isolate is due to
production of extracellular peroxidase-type enzymes and glycosylated
haem-based peroxidase (DyP) (Kim and Shoda 1999a)
However application of white-rot fungi for the removal of dyes from
textile wastewater have inherent drawbacks long growth cycle requiring
nitrogen limiting conditions naturally white rot fungi not found in wastewater
hence the enzyme production may be unreliable (Robinson et al 2001) long
hydraulic retention time for complete decolorization still limit the performance
of the fungal decolorization system (Banat et al 1996 Saratale et al 2009b) as
well as preservation in bioreactors will be a matter of concern (Stolz 2001)
214 Decolorization with yeast
Very little work was devoted to exploring the decolorization ability of
yeast mainly studied for the biosorption It was observed that few ascomycetes
yeast species such as Candida tropicalis Debaryomyces polymorphus (Yang et
al 2003) Candida zeylanoides (Martins et al 1999 Ramalho et al 2002) and
Issatchenkia occidentalis (Ramalho et al 2004) perform a putative enzymatic
biodegradation and consequent decolorization of different azo dyes Earlier
Saccharomyces cerevisiae MTCC-463 was reported to involve in the
decolorization of Malachite Green and Methyl Red (Jadhav et al 2006 Jadhav
et al 2007) S cerevisiae cells also showed bioaccumulation of reactive textile
dye (Remazol Blue Remazol Black B and Remazol Red RB) during growth in
molasses (Aksu 2003) Magnetically modified bakers yeast has been used for
the biosorption of Acridine Orange Aniline Blue Crystal Violet Malachite
Green and Safranine acts as a promising dye adsorbent (ˇSafaˇrikova et al
2005) Biological decolorization of triphenylmethane dyes are widely reported
using red yeasts Rhodotorulae sp and Rhodotorulae rubra (Kwasniewska 1995)
19
Review of literature
Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane azo
and reactive high exhaust textile dyes as reported earlier (Jadhav et al 2008)
Recently detailed study on the decolorization of Navy Blue HER by using
Trichosporon beigelii NCIM-3326 along with the enzymatic mechanism and
toxicity of degradation products has been reported (Saratale et al 2009a) In a
comparative study on biosorption capacities of different kinds of dried yeasts for
Remazol Blue Candida lipolytica showed the highest dye uptake capacity up to
250 mg g-1 cells (Donmez 2002 Aksu and Donmez 2003)
215 Decolorization with algae
The photosynthetic organisms such as cyanobacteria or algae have a
ubiquitous distribution and observed in all kind of habitats of the world The
literature survey suggests that algae are capable of degrading azo dyes through
an induced form of an azoreductase (Jinqi and Houtian 1992) Color removal by
algae was due to three intrinsically different mechanisms of assimilative
utilization of chromophores for production of algal biomass CO2 and H2O
transformation of colored molecules to non-colored molecules and adsorption of
chromophores on algal biomass Several species of Chlorella (Acuner and Dilek
2004) and Oscillatoria (Jinqi and Houtian 1992) were capable of degrading azo
dyes to their aromatic amines and to further metabolize the aromatic amines to
simpler organic compounds or CO2 Mohan et al (2002) attributes the
decolorization to biosorption followed by bioconversion and biocoagulation It
was reported that more than 30 azo compounds were biodegraded and
decolorized by Chlorella pyrenoidosa Chlorella vulgaris and Oscillateria
tenuis in which azo dyes were decomposed into simpler aromatic amines (Yan
and Pan 2004) Thus the foregoing results could mean that algae can play an
20
Review of literature
important role in the removal of azo dyes and aromatic amines in stabilization
ponds (Banat et al 1996)
216 Decolorization with plant (phytoremediation)
Phytoremediation is considered as a plausible approach for the
remediation of soils and groundwater contaminated with heavy metals and
organic pollutants Recently some studies describe the use of plants for the dye
removal from wastewaters The Rheum rabarbarum mentions a good removal
capacity of sulfonated anthraquinones (Aubert and Schwitzgueacutebel 2004)
Recently narrow-leaved cattails were studied in synthetic reactive dye
wastewater treatment under caustic conditions (Nilratnisakorn et al 2007) Also
Mbuligwe describes a reduction in color of 72-77 in wetlands vegetated with
coco yam plants (Mbuligwe 2005) It was reported that the plant possesses
enzymes that accept anthraquinones as substrates and in cell culture were able to
remove up to 700-800 mg l-1 of anthraquinones with sulphonate groups in
different positions The three plant species (Brassica juncea Sorghum vulgare
and Phaseolus mungo) of different agronomic consequence were evaluated for
the decolorization of the dyes from textile effluent These plants B juncea S
vulgare and P mungo showed textile effluent decolorization up to 79 57 and
53 respectively (Ghodake et al 2008) Similarly an herb Blumea malcommi
was found to degrade textile dye Reactive Red 5B (Kagalkar et al 2009)
However in large scale application of phytoremediation presently faces a
number of obstacles including the level of pollutants tolerated by the plant the
bioavailable fraction of the contaminants and evapotranspiration of volatile
organic pollutants as well as requiring big areas to implant the treatment
(Williams 2002)
217 Bacterial decolorization and degradation of azo dyes
21
Review of literature
Generally the decolorization of azo dyes occurs under conventional
anaerobic anoxic and aerobic conditions by different groups of the bacteria The
mechanism of microbial degradation of azo dyes involves the reductive cleavage
of azo bonds (-N=N-) with the help of azoreductase under anaerobic conditions
resulted into the formation of colorless solutions containing potentially
hazardous-aromatic amines (Chang et al 2000 van der Zee and Villaverde
2005) The resulting intermediate metabolites (eg aromatic amines) are further
degraded aerobically or anaerobically (Seshadri et al 1994) Many recent
researches focus on utilization of microbial biocatalyst to reduce the dye from
the effluent (Hu 1998 Chang et al 2000 Martina Mohorčič 2004) Moreover
the effectiveness of microbial decolorization depends on the adaptability and the
activity of selected microorganisms It has been reported that a wide range of
microorganisms including bacteria fungi yeasts actinomycetes and algae are
capable of degrading azo dyes Moreover most studies on azo dye
biodegradation have focused on the bacteria and fungi The fungal cultures
mainly belonging to white rot fungi have been used to develop bioprocesses for
the mineralization of azo dyes (Parshetti et al 2007) However a long growth
cycle requiring nitrogen limiting conditions and long hydraulic retention time
for complete decolorization still limit the performance of the fungal
decolorization system (Banat et al 1996 Chang et al 2004 Jadhav et al
2008) In contrast bacterial decolorization and degradation of azo dyes has been
of considerable interest since it possesses higher degree of biodegradation and
mineralization diversity towards variety of azo dyes inexpensive and
eco-friendly nature and less sludge producing properties (Verma and Madmawar
2003 Rai et al 2005 Saratale et al 2009c Kalyani et al 2008 Dhanve et al
2008 Saratale et al 2006) Extensive studies have been carried out to determine
22
Review of literature
the role of the diverse groups of bacteria in the decolorization of azo dyes
(Pandey et al 2007)
i) Using pure bacterial culture
The effluents from textile industries are complex containing a wide
variety of dyes and other products such as dispersants acids bases salts
detergents humectants oxidants etc Discharge of these colored effluents into
rivers and lakes results into reduced dissolved oxygen concentration thus
creating anoxic conditions that are lethal to resident organisms Biological
processes provide an alternative to existing technologies because they are more
cost-effective environmentally friendly and do not produce large quantities of
sludge Bacterial decolorization is normally faster compared to fungal system for
the decolorization and mineralization of azo dyes It was observed that the mixed
cultures are apparent as some microbial consortia can collectively carry out
biodegradation tasks that no individual pure strain can undertake successfully
(Nigam et al 1996) However mixed cultures only provide an average
macroscopic view of what is happening in the system and results are not easily
reproduced making thorough effective interpretation difficult For these
reasons a substantial amount of research on the subject of color removal has been
carried out using single bacterial cultures like P mirabilis P luteola
Pseudomonas sp Pseudomonas sp SUK1 and K rosea has shown very
promising results for the dye degradation under anoxic conditions (Chen et al
1999 Chang et al 2001 Yu et al 2001 Kalyani et al 2008 Parshetti et al
2006) In addition there are also several studies describing the decolorization of
reactive dyes mediated by pure bacterial culture such as Exiguobacterium sp
RD3 for Navy Blue HE2R (Reactive Blue 172) (Dhanve et al 2008) Rhizobium
radiobacter MTCC 8161 for Reactive Red 141 (Telke et al 2008) P vulgaris
23
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
eco-friendly nature 2) cost-competitive 3) less sludge producing properties 4)
the end products with complete mineralization or non toxic products and 5)
could help to reduce the enormous water consumption compared to
physicochemical methods (Saratale et al 2009b Rai et al 2005) Moreover the
effectiveness of the microbial decolorization depends upon the adaptability and
the activity of selected microorganisms Large number of species has been tested
for the decolorization and mineralization of various dyes and steadily increasing
in recent years (Pandey et al 2007) The isolation of potent species and there by
degradation is one of the interest in biological aspect of effluents treatment
(Mohan et al 2002) A wide variety of microorganisms are capable of
decolorization of a wide range of dyes using wide range of microorganisms
including bacteria (Parshetti et al 2006 Kalyani et al 2008 Telke et al 2008
Dawkar et al 2008 Saratale et al 2009c Jadhav et al 2009) fungi (Fournier
et al 2004 Saratale et al2006 Machado et al 2006 Humnabadkar et al
2008) yeasts (Lucas et al 2006 Jadhav et al 2007 Saratale et al 2009a)
actinomycetes (Parshetti et al 2007) algae (Acuner and Dilek 2004 Yan and
Pan 2004 Parikh and Madamwar 2005 Gupta et al 2006 Daneshvar et al
2007) and plants (Phytoremediation) (Aubert and Schwitzguebel 2004
Ghodake et al 2009 Kagalkar et al 2009) capable of decolorize and even
completely mineralize many azo dyes under certain environmental conditions
213 Fungal decolorization and degradation of textile dyes
Filamentous fungi are found ubiquitous in the environment inhabiting
ecological niches such as soil living plants and organic waste material The
ability of fungi to rapidly adapt their metabolism to varying carbon and nitrogen
sources is an integrated aspect for their survival This metabolic activity
achieved through the production of a large set of intra and extracellular enzymes
17
Review of literature
able to degrade complex various kinds of organic pollutants (Saratale et al
2007a) In addition to the production and secretion of number of enzymes
filamentous fungi can secrete a great diversity of primary and secondary
metabolites (eg antibiotics) and perform many different complex conversions
such as hydroxylation of complex polyaromatic hydrocarbons organic waste
dye effluents and steroid compounds (McMullan et al 2001 Saratale et al
2007b) Fungal systems appear to be the most appropriate in the treatment of
colored and metallic effluents (Ezeronye and Okerentugba 1999) The ability of
these fungi to degrade such a range of organic compounds results from the
relatively non-specific nature of their ligninolytic enzymes such as lignin
peroxidase (LiP) manganese peroxidase (MnP) and laccase Fungal degradation
of aromatic structures is a secondary metabolic event that starts when nutrients
(C N and S) become limiting (Kirk and Farrell 1987) Therefore while the
enzymes are optimally expressed under starving conditions supplementation of
energy substrates and nutrients are necessary for the propagation of the cultures
(Christian et al 2005)
Most studies on an azo dye biodegradation have focused on the fungal
cultures mainly belonging to white rot fungi and used to develop bioprocesses
for mineralization of azo dyes (Parshetti et al 2007) The Phanerochaete
chrysosporium is the most widely studied of white-rot fungi as well as Trametes
(Coriolus) versicolor Bjerkandera adusta Pleurotus Aspergillus and Phlebia
species and variety of other isolates also studied for the degradation of various
textile dyes (Heinfling et al 1998 Conneely et al 1999 Swamy and Ramsay
1999 Pointing et al 2000 Saratale et al 2006 Humnabadkar et al 2008) A
detailed investigation was also carried out on isolated Geotrichum candidum
Dec1 capable of decolorization a number of anthraquinone dyes (Kim et al
18
Review of literature
1995) The broad substrate specificity exhibited by this isolate is due to
production of extracellular peroxidase-type enzymes and glycosylated
haem-based peroxidase (DyP) (Kim and Shoda 1999a)
However application of white-rot fungi for the removal of dyes from
textile wastewater have inherent drawbacks long growth cycle requiring
nitrogen limiting conditions naturally white rot fungi not found in wastewater
hence the enzyme production may be unreliable (Robinson et al 2001) long
hydraulic retention time for complete decolorization still limit the performance
of the fungal decolorization system (Banat et al 1996 Saratale et al 2009b) as
well as preservation in bioreactors will be a matter of concern (Stolz 2001)
214 Decolorization with yeast
Very little work was devoted to exploring the decolorization ability of
yeast mainly studied for the biosorption It was observed that few ascomycetes
yeast species such as Candida tropicalis Debaryomyces polymorphus (Yang et
al 2003) Candida zeylanoides (Martins et al 1999 Ramalho et al 2002) and
Issatchenkia occidentalis (Ramalho et al 2004) perform a putative enzymatic
biodegradation and consequent decolorization of different azo dyes Earlier
Saccharomyces cerevisiae MTCC-463 was reported to involve in the
decolorization of Malachite Green and Methyl Red (Jadhav et al 2006 Jadhav
et al 2007) S cerevisiae cells also showed bioaccumulation of reactive textile
dye (Remazol Blue Remazol Black B and Remazol Red RB) during growth in
molasses (Aksu 2003) Magnetically modified bakers yeast has been used for
the biosorption of Acridine Orange Aniline Blue Crystal Violet Malachite
Green and Safranine acts as a promising dye adsorbent (ˇSafaˇrikova et al
2005) Biological decolorization of triphenylmethane dyes are widely reported
using red yeasts Rhodotorulae sp and Rhodotorulae rubra (Kwasniewska 1995)
19
Review of literature
Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane azo
and reactive high exhaust textile dyes as reported earlier (Jadhav et al 2008)
Recently detailed study on the decolorization of Navy Blue HER by using
Trichosporon beigelii NCIM-3326 along with the enzymatic mechanism and
toxicity of degradation products has been reported (Saratale et al 2009a) In a
comparative study on biosorption capacities of different kinds of dried yeasts for
Remazol Blue Candida lipolytica showed the highest dye uptake capacity up to
250 mg g-1 cells (Donmez 2002 Aksu and Donmez 2003)
215 Decolorization with algae
The photosynthetic organisms such as cyanobacteria or algae have a
ubiquitous distribution and observed in all kind of habitats of the world The
literature survey suggests that algae are capable of degrading azo dyes through
an induced form of an azoreductase (Jinqi and Houtian 1992) Color removal by
algae was due to three intrinsically different mechanisms of assimilative
utilization of chromophores for production of algal biomass CO2 and H2O
transformation of colored molecules to non-colored molecules and adsorption of
chromophores on algal biomass Several species of Chlorella (Acuner and Dilek
2004) and Oscillatoria (Jinqi and Houtian 1992) were capable of degrading azo
dyes to their aromatic amines and to further metabolize the aromatic amines to
simpler organic compounds or CO2 Mohan et al (2002) attributes the
decolorization to biosorption followed by bioconversion and biocoagulation It
was reported that more than 30 azo compounds were biodegraded and
decolorized by Chlorella pyrenoidosa Chlorella vulgaris and Oscillateria
tenuis in which azo dyes were decomposed into simpler aromatic amines (Yan
and Pan 2004) Thus the foregoing results could mean that algae can play an
20
Review of literature
important role in the removal of azo dyes and aromatic amines in stabilization
ponds (Banat et al 1996)
216 Decolorization with plant (phytoremediation)
Phytoremediation is considered as a plausible approach for the
remediation of soils and groundwater contaminated with heavy metals and
organic pollutants Recently some studies describe the use of plants for the dye
removal from wastewaters The Rheum rabarbarum mentions a good removal
capacity of sulfonated anthraquinones (Aubert and Schwitzgueacutebel 2004)
Recently narrow-leaved cattails were studied in synthetic reactive dye
wastewater treatment under caustic conditions (Nilratnisakorn et al 2007) Also
Mbuligwe describes a reduction in color of 72-77 in wetlands vegetated with
coco yam plants (Mbuligwe 2005) It was reported that the plant possesses
enzymes that accept anthraquinones as substrates and in cell culture were able to
remove up to 700-800 mg l-1 of anthraquinones with sulphonate groups in
different positions The three plant species (Brassica juncea Sorghum vulgare
and Phaseolus mungo) of different agronomic consequence were evaluated for
the decolorization of the dyes from textile effluent These plants B juncea S
vulgare and P mungo showed textile effluent decolorization up to 79 57 and
53 respectively (Ghodake et al 2008) Similarly an herb Blumea malcommi
was found to degrade textile dye Reactive Red 5B (Kagalkar et al 2009)
However in large scale application of phytoremediation presently faces a
number of obstacles including the level of pollutants tolerated by the plant the
bioavailable fraction of the contaminants and evapotranspiration of volatile
organic pollutants as well as requiring big areas to implant the treatment
(Williams 2002)
217 Bacterial decolorization and degradation of azo dyes
21
Review of literature
Generally the decolorization of azo dyes occurs under conventional
anaerobic anoxic and aerobic conditions by different groups of the bacteria The
mechanism of microbial degradation of azo dyes involves the reductive cleavage
of azo bonds (-N=N-) with the help of azoreductase under anaerobic conditions
resulted into the formation of colorless solutions containing potentially
hazardous-aromatic amines (Chang et al 2000 van der Zee and Villaverde
2005) The resulting intermediate metabolites (eg aromatic amines) are further
degraded aerobically or anaerobically (Seshadri et al 1994) Many recent
researches focus on utilization of microbial biocatalyst to reduce the dye from
the effluent (Hu 1998 Chang et al 2000 Martina Mohorčič 2004) Moreover
the effectiveness of microbial decolorization depends on the adaptability and the
activity of selected microorganisms It has been reported that a wide range of
microorganisms including bacteria fungi yeasts actinomycetes and algae are
capable of degrading azo dyes Moreover most studies on azo dye
biodegradation have focused on the bacteria and fungi The fungal cultures
mainly belonging to white rot fungi have been used to develop bioprocesses for
the mineralization of azo dyes (Parshetti et al 2007) However a long growth
cycle requiring nitrogen limiting conditions and long hydraulic retention time
for complete decolorization still limit the performance of the fungal
decolorization system (Banat et al 1996 Chang et al 2004 Jadhav et al
2008) In contrast bacterial decolorization and degradation of azo dyes has been
of considerable interest since it possesses higher degree of biodegradation and
mineralization diversity towards variety of azo dyes inexpensive and
eco-friendly nature and less sludge producing properties (Verma and Madmawar
2003 Rai et al 2005 Saratale et al 2009c Kalyani et al 2008 Dhanve et al
2008 Saratale et al 2006) Extensive studies have been carried out to determine
22
Review of literature
the role of the diverse groups of bacteria in the decolorization of azo dyes
(Pandey et al 2007)
i) Using pure bacterial culture
The effluents from textile industries are complex containing a wide
variety of dyes and other products such as dispersants acids bases salts
detergents humectants oxidants etc Discharge of these colored effluents into
rivers and lakes results into reduced dissolved oxygen concentration thus
creating anoxic conditions that are lethal to resident organisms Biological
processes provide an alternative to existing technologies because they are more
cost-effective environmentally friendly and do not produce large quantities of
sludge Bacterial decolorization is normally faster compared to fungal system for
the decolorization and mineralization of azo dyes It was observed that the mixed
cultures are apparent as some microbial consortia can collectively carry out
biodegradation tasks that no individual pure strain can undertake successfully
(Nigam et al 1996) However mixed cultures only provide an average
macroscopic view of what is happening in the system and results are not easily
reproduced making thorough effective interpretation difficult For these
reasons a substantial amount of research on the subject of color removal has been
carried out using single bacterial cultures like P mirabilis P luteola
Pseudomonas sp Pseudomonas sp SUK1 and K rosea has shown very
promising results for the dye degradation under anoxic conditions (Chen et al
1999 Chang et al 2001 Yu et al 2001 Kalyani et al 2008 Parshetti et al
2006) In addition there are also several studies describing the decolorization of
reactive dyes mediated by pure bacterial culture such as Exiguobacterium sp
RD3 for Navy Blue HE2R (Reactive Blue 172) (Dhanve et al 2008) Rhizobium
radiobacter MTCC 8161 for Reactive Red 141 (Telke et al 2008) P vulgaris
23
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
able to degrade complex various kinds of organic pollutants (Saratale et al
2007a) In addition to the production and secretion of number of enzymes
filamentous fungi can secrete a great diversity of primary and secondary
metabolites (eg antibiotics) and perform many different complex conversions
such as hydroxylation of complex polyaromatic hydrocarbons organic waste
dye effluents and steroid compounds (McMullan et al 2001 Saratale et al
2007b) Fungal systems appear to be the most appropriate in the treatment of
colored and metallic effluents (Ezeronye and Okerentugba 1999) The ability of
these fungi to degrade such a range of organic compounds results from the
relatively non-specific nature of their ligninolytic enzymes such as lignin
peroxidase (LiP) manganese peroxidase (MnP) and laccase Fungal degradation
of aromatic structures is a secondary metabolic event that starts when nutrients
(C N and S) become limiting (Kirk and Farrell 1987) Therefore while the
enzymes are optimally expressed under starving conditions supplementation of
energy substrates and nutrients are necessary for the propagation of the cultures
(Christian et al 2005)
Most studies on an azo dye biodegradation have focused on the fungal
cultures mainly belonging to white rot fungi and used to develop bioprocesses
for mineralization of azo dyes (Parshetti et al 2007) The Phanerochaete
chrysosporium is the most widely studied of white-rot fungi as well as Trametes
(Coriolus) versicolor Bjerkandera adusta Pleurotus Aspergillus and Phlebia
species and variety of other isolates also studied for the degradation of various
textile dyes (Heinfling et al 1998 Conneely et al 1999 Swamy and Ramsay
1999 Pointing et al 2000 Saratale et al 2006 Humnabadkar et al 2008) A
detailed investigation was also carried out on isolated Geotrichum candidum
Dec1 capable of decolorization a number of anthraquinone dyes (Kim et al
18
Review of literature
1995) The broad substrate specificity exhibited by this isolate is due to
production of extracellular peroxidase-type enzymes and glycosylated
haem-based peroxidase (DyP) (Kim and Shoda 1999a)
However application of white-rot fungi for the removal of dyes from
textile wastewater have inherent drawbacks long growth cycle requiring
nitrogen limiting conditions naturally white rot fungi not found in wastewater
hence the enzyme production may be unreliable (Robinson et al 2001) long
hydraulic retention time for complete decolorization still limit the performance
of the fungal decolorization system (Banat et al 1996 Saratale et al 2009b) as
well as preservation in bioreactors will be a matter of concern (Stolz 2001)
214 Decolorization with yeast
Very little work was devoted to exploring the decolorization ability of
yeast mainly studied for the biosorption It was observed that few ascomycetes
yeast species such as Candida tropicalis Debaryomyces polymorphus (Yang et
al 2003) Candida zeylanoides (Martins et al 1999 Ramalho et al 2002) and
Issatchenkia occidentalis (Ramalho et al 2004) perform a putative enzymatic
biodegradation and consequent decolorization of different azo dyes Earlier
Saccharomyces cerevisiae MTCC-463 was reported to involve in the
decolorization of Malachite Green and Methyl Red (Jadhav et al 2006 Jadhav
et al 2007) S cerevisiae cells also showed bioaccumulation of reactive textile
dye (Remazol Blue Remazol Black B and Remazol Red RB) during growth in
molasses (Aksu 2003) Magnetically modified bakers yeast has been used for
the biosorption of Acridine Orange Aniline Blue Crystal Violet Malachite
Green and Safranine acts as a promising dye adsorbent (ˇSafaˇrikova et al
2005) Biological decolorization of triphenylmethane dyes are widely reported
using red yeasts Rhodotorulae sp and Rhodotorulae rubra (Kwasniewska 1995)
19
Review of literature
Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane azo
and reactive high exhaust textile dyes as reported earlier (Jadhav et al 2008)
Recently detailed study on the decolorization of Navy Blue HER by using
Trichosporon beigelii NCIM-3326 along with the enzymatic mechanism and
toxicity of degradation products has been reported (Saratale et al 2009a) In a
comparative study on biosorption capacities of different kinds of dried yeasts for
Remazol Blue Candida lipolytica showed the highest dye uptake capacity up to
250 mg g-1 cells (Donmez 2002 Aksu and Donmez 2003)
215 Decolorization with algae
The photosynthetic organisms such as cyanobacteria or algae have a
ubiquitous distribution and observed in all kind of habitats of the world The
literature survey suggests that algae are capable of degrading azo dyes through
an induced form of an azoreductase (Jinqi and Houtian 1992) Color removal by
algae was due to three intrinsically different mechanisms of assimilative
utilization of chromophores for production of algal biomass CO2 and H2O
transformation of colored molecules to non-colored molecules and adsorption of
chromophores on algal biomass Several species of Chlorella (Acuner and Dilek
2004) and Oscillatoria (Jinqi and Houtian 1992) were capable of degrading azo
dyes to their aromatic amines and to further metabolize the aromatic amines to
simpler organic compounds or CO2 Mohan et al (2002) attributes the
decolorization to biosorption followed by bioconversion and biocoagulation It
was reported that more than 30 azo compounds were biodegraded and
decolorized by Chlorella pyrenoidosa Chlorella vulgaris and Oscillateria
tenuis in which azo dyes were decomposed into simpler aromatic amines (Yan
and Pan 2004) Thus the foregoing results could mean that algae can play an
20
Review of literature
important role in the removal of azo dyes and aromatic amines in stabilization
ponds (Banat et al 1996)
216 Decolorization with plant (phytoremediation)
Phytoremediation is considered as a plausible approach for the
remediation of soils and groundwater contaminated with heavy metals and
organic pollutants Recently some studies describe the use of plants for the dye
removal from wastewaters The Rheum rabarbarum mentions a good removal
capacity of sulfonated anthraquinones (Aubert and Schwitzgueacutebel 2004)
Recently narrow-leaved cattails were studied in synthetic reactive dye
wastewater treatment under caustic conditions (Nilratnisakorn et al 2007) Also
Mbuligwe describes a reduction in color of 72-77 in wetlands vegetated with
coco yam plants (Mbuligwe 2005) It was reported that the plant possesses
enzymes that accept anthraquinones as substrates and in cell culture were able to
remove up to 700-800 mg l-1 of anthraquinones with sulphonate groups in
different positions The three plant species (Brassica juncea Sorghum vulgare
and Phaseolus mungo) of different agronomic consequence were evaluated for
the decolorization of the dyes from textile effluent These plants B juncea S
vulgare and P mungo showed textile effluent decolorization up to 79 57 and
53 respectively (Ghodake et al 2008) Similarly an herb Blumea malcommi
was found to degrade textile dye Reactive Red 5B (Kagalkar et al 2009)
However in large scale application of phytoremediation presently faces a
number of obstacles including the level of pollutants tolerated by the plant the
bioavailable fraction of the contaminants and evapotranspiration of volatile
organic pollutants as well as requiring big areas to implant the treatment
(Williams 2002)
217 Bacterial decolorization and degradation of azo dyes
21
Review of literature
Generally the decolorization of azo dyes occurs under conventional
anaerobic anoxic and aerobic conditions by different groups of the bacteria The
mechanism of microbial degradation of azo dyes involves the reductive cleavage
of azo bonds (-N=N-) with the help of azoreductase under anaerobic conditions
resulted into the formation of colorless solutions containing potentially
hazardous-aromatic amines (Chang et al 2000 van der Zee and Villaverde
2005) The resulting intermediate metabolites (eg aromatic amines) are further
degraded aerobically or anaerobically (Seshadri et al 1994) Many recent
researches focus on utilization of microbial biocatalyst to reduce the dye from
the effluent (Hu 1998 Chang et al 2000 Martina Mohorčič 2004) Moreover
the effectiveness of microbial decolorization depends on the adaptability and the
activity of selected microorganisms It has been reported that a wide range of
microorganisms including bacteria fungi yeasts actinomycetes and algae are
capable of degrading azo dyes Moreover most studies on azo dye
biodegradation have focused on the bacteria and fungi The fungal cultures
mainly belonging to white rot fungi have been used to develop bioprocesses for
the mineralization of azo dyes (Parshetti et al 2007) However a long growth
cycle requiring nitrogen limiting conditions and long hydraulic retention time
for complete decolorization still limit the performance of the fungal
decolorization system (Banat et al 1996 Chang et al 2004 Jadhav et al
2008) In contrast bacterial decolorization and degradation of azo dyes has been
of considerable interest since it possesses higher degree of biodegradation and
mineralization diversity towards variety of azo dyes inexpensive and
eco-friendly nature and less sludge producing properties (Verma and Madmawar
2003 Rai et al 2005 Saratale et al 2009c Kalyani et al 2008 Dhanve et al
2008 Saratale et al 2006) Extensive studies have been carried out to determine
22
Review of literature
the role of the diverse groups of bacteria in the decolorization of azo dyes
(Pandey et al 2007)
i) Using pure bacterial culture
The effluents from textile industries are complex containing a wide
variety of dyes and other products such as dispersants acids bases salts
detergents humectants oxidants etc Discharge of these colored effluents into
rivers and lakes results into reduced dissolved oxygen concentration thus
creating anoxic conditions that are lethal to resident organisms Biological
processes provide an alternative to existing technologies because they are more
cost-effective environmentally friendly and do not produce large quantities of
sludge Bacterial decolorization is normally faster compared to fungal system for
the decolorization and mineralization of azo dyes It was observed that the mixed
cultures are apparent as some microbial consortia can collectively carry out
biodegradation tasks that no individual pure strain can undertake successfully
(Nigam et al 1996) However mixed cultures only provide an average
macroscopic view of what is happening in the system and results are not easily
reproduced making thorough effective interpretation difficult For these
reasons a substantial amount of research on the subject of color removal has been
carried out using single bacterial cultures like P mirabilis P luteola
Pseudomonas sp Pseudomonas sp SUK1 and K rosea has shown very
promising results for the dye degradation under anoxic conditions (Chen et al
1999 Chang et al 2001 Yu et al 2001 Kalyani et al 2008 Parshetti et al
2006) In addition there are also several studies describing the decolorization of
reactive dyes mediated by pure bacterial culture such as Exiguobacterium sp
RD3 for Navy Blue HE2R (Reactive Blue 172) (Dhanve et al 2008) Rhizobium
radiobacter MTCC 8161 for Reactive Red 141 (Telke et al 2008) P vulgaris
23
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
1995) The broad substrate specificity exhibited by this isolate is due to
production of extracellular peroxidase-type enzymes and glycosylated
haem-based peroxidase (DyP) (Kim and Shoda 1999a)
However application of white-rot fungi for the removal of dyes from
textile wastewater have inherent drawbacks long growth cycle requiring
nitrogen limiting conditions naturally white rot fungi not found in wastewater
hence the enzyme production may be unreliable (Robinson et al 2001) long
hydraulic retention time for complete decolorization still limit the performance
of the fungal decolorization system (Banat et al 1996 Saratale et al 2009b) as
well as preservation in bioreactors will be a matter of concern (Stolz 2001)
214 Decolorization with yeast
Very little work was devoted to exploring the decolorization ability of
yeast mainly studied for the biosorption It was observed that few ascomycetes
yeast species such as Candida tropicalis Debaryomyces polymorphus (Yang et
al 2003) Candida zeylanoides (Martins et al 1999 Ramalho et al 2002) and
Issatchenkia occidentalis (Ramalho et al 2004) perform a putative enzymatic
biodegradation and consequent decolorization of different azo dyes Earlier
Saccharomyces cerevisiae MTCC-463 was reported to involve in the
decolorization of Malachite Green and Methyl Red (Jadhav et al 2006 Jadhav
et al 2007) S cerevisiae cells also showed bioaccumulation of reactive textile
dye (Remazol Blue Remazol Black B and Remazol Red RB) during growth in
molasses (Aksu 2003) Magnetically modified bakers yeast has been used for
the biosorption of Acridine Orange Aniline Blue Crystal Violet Malachite
Green and Safranine acts as a promising dye adsorbent (ˇSafaˇrikova et al
2005) Biological decolorization of triphenylmethane dyes are widely reported
using red yeasts Rhodotorulae sp and Rhodotorulae rubra (Kwasniewska 1995)
19
Review of literature
Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane azo
and reactive high exhaust textile dyes as reported earlier (Jadhav et al 2008)
Recently detailed study on the decolorization of Navy Blue HER by using
Trichosporon beigelii NCIM-3326 along with the enzymatic mechanism and
toxicity of degradation products has been reported (Saratale et al 2009a) In a
comparative study on biosorption capacities of different kinds of dried yeasts for
Remazol Blue Candida lipolytica showed the highest dye uptake capacity up to
250 mg g-1 cells (Donmez 2002 Aksu and Donmez 2003)
215 Decolorization with algae
The photosynthetic organisms such as cyanobacteria or algae have a
ubiquitous distribution and observed in all kind of habitats of the world The
literature survey suggests that algae are capable of degrading azo dyes through
an induced form of an azoreductase (Jinqi and Houtian 1992) Color removal by
algae was due to three intrinsically different mechanisms of assimilative
utilization of chromophores for production of algal biomass CO2 and H2O
transformation of colored molecules to non-colored molecules and adsorption of
chromophores on algal biomass Several species of Chlorella (Acuner and Dilek
2004) and Oscillatoria (Jinqi and Houtian 1992) were capable of degrading azo
dyes to their aromatic amines and to further metabolize the aromatic amines to
simpler organic compounds or CO2 Mohan et al (2002) attributes the
decolorization to biosorption followed by bioconversion and biocoagulation It
was reported that more than 30 azo compounds were biodegraded and
decolorized by Chlorella pyrenoidosa Chlorella vulgaris and Oscillateria
tenuis in which azo dyes were decomposed into simpler aromatic amines (Yan
and Pan 2004) Thus the foregoing results could mean that algae can play an
20
Review of literature
important role in the removal of azo dyes and aromatic amines in stabilization
ponds (Banat et al 1996)
216 Decolorization with plant (phytoremediation)
Phytoremediation is considered as a plausible approach for the
remediation of soils and groundwater contaminated with heavy metals and
organic pollutants Recently some studies describe the use of plants for the dye
removal from wastewaters The Rheum rabarbarum mentions a good removal
capacity of sulfonated anthraquinones (Aubert and Schwitzgueacutebel 2004)
Recently narrow-leaved cattails were studied in synthetic reactive dye
wastewater treatment under caustic conditions (Nilratnisakorn et al 2007) Also
Mbuligwe describes a reduction in color of 72-77 in wetlands vegetated with
coco yam plants (Mbuligwe 2005) It was reported that the plant possesses
enzymes that accept anthraquinones as substrates and in cell culture were able to
remove up to 700-800 mg l-1 of anthraquinones with sulphonate groups in
different positions The three plant species (Brassica juncea Sorghum vulgare
and Phaseolus mungo) of different agronomic consequence were evaluated for
the decolorization of the dyes from textile effluent These plants B juncea S
vulgare and P mungo showed textile effluent decolorization up to 79 57 and
53 respectively (Ghodake et al 2008) Similarly an herb Blumea malcommi
was found to degrade textile dye Reactive Red 5B (Kagalkar et al 2009)
However in large scale application of phytoremediation presently faces a
number of obstacles including the level of pollutants tolerated by the plant the
bioavailable fraction of the contaminants and evapotranspiration of volatile
organic pollutants as well as requiring big areas to implant the treatment
(Williams 2002)
217 Bacterial decolorization and degradation of azo dyes
21
Review of literature
Generally the decolorization of azo dyes occurs under conventional
anaerobic anoxic and aerobic conditions by different groups of the bacteria The
mechanism of microbial degradation of azo dyes involves the reductive cleavage
of azo bonds (-N=N-) with the help of azoreductase under anaerobic conditions
resulted into the formation of colorless solutions containing potentially
hazardous-aromatic amines (Chang et al 2000 van der Zee and Villaverde
2005) The resulting intermediate metabolites (eg aromatic amines) are further
degraded aerobically or anaerobically (Seshadri et al 1994) Many recent
researches focus on utilization of microbial biocatalyst to reduce the dye from
the effluent (Hu 1998 Chang et al 2000 Martina Mohorčič 2004) Moreover
the effectiveness of microbial decolorization depends on the adaptability and the
activity of selected microorganisms It has been reported that a wide range of
microorganisms including bacteria fungi yeasts actinomycetes and algae are
capable of degrading azo dyes Moreover most studies on azo dye
biodegradation have focused on the bacteria and fungi The fungal cultures
mainly belonging to white rot fungi have been used to develop bioprocesses for
the mineralization of azo dyes (Parshetti et al 2007) However a long growth
cycle requiring nitrogen limiting conditions and long hydraulic retention time
for complete decolorization still limit the performance of the fungal
decolorization system (Banat et al 1996 Chang et al 2004 Jadhav et al
2008) In contrast bacterial decolorization and degradation of azo dyes has been
of considerable interest since it possesses higher degree of biodegradation and
mineralization diversity towards variety of azo dyes inexpensive and
eco-friendly nature and less sludge producing properties (Verma and Madmawar
2003 Rai et al 2005 Saratale et al 2009c Kalyani et al 2008 Dhanve et al
2008 Saratale et al 2006) Extensive studies have been carried out to determine
22
Review of literature
the role of the diverse groups of bacteria in the decolorization of azo dyes
(Pandey et al 2007)
i) Using pure bacterial culture
The effluents from textile industries are complex containing a wide
variety of dyes and other products such as dispersants acids bases salts
detergents humectants oxidants etc Discharge of these colored effluents into
rivers and lakes results into reduced dissolved oxygen concentration thus
creating anoxic conditions that are lethal to resident organisms Biological
processes provide an alternative to existing technologies because they are more
cost-effective environmentally friendly and do not produce large quantities of
sludge Bacterial decolorization is normally faster compared to fungal system for
the decolorization and mineralization of azo dyes It was observed that the mixed
cultures are apparent as some microbial consortia can collectively carry out
biodegradation tasks that no individual pure strain can undertake successfully
(Nigam et al 1996) However mixed cultures only provide an average
macroscopic view of what is happening in the system and results are not easily
reproduced making thorough effective interpretation difficult For these
reasons a substantial amount of research on the subject of color removal has been
carried out using single bacterial cultures like P mirabilis P luteola
Pseudomonas sp Pseudomonas sp SUK1 and K rosea has shown very
promising results for the dye degradation under anoxic conditions (Chen et al
1999 Chang et al 2001 Yu et al 2001 Kalyani et al 2008 Parshetti et al
2006) In addition there are also several studies describing the decolorization of
reactive dyes mediated by pure bacterial culture such as Exiguobacterium sp
RD3 for Navy Blue HE2R (Reactive Blue 172) (Dhanve et al 2008) Rhizobium
radiobacter MTCC 8161 for Reactive Red 141 (Telke et al 2008) P vulgaris
23
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane azo
and reactive high exhaust textile dyes as reported earlier (Jadhav et al 2008)
Recently detailed study on the decolorization of Navy Blue HER by using
Trichosporon beigelii NCIM-3326 along with the enzymatic mechanism and
toxicity of degradation products has been reported (Saratale et al 2009a) In a
comparative study on biosorption capacities of different kinds of dried yeasts for
Remazol Blue Candida lipolytica showed the highest dye uptake capacity up to
250 mg g-1 cells (Donmez 2002 Aksu and Donmez 2003)
215 Decolorization with algae
The photosynthetic organisms such as cyanobacteria or algae have a
ubiquitous distribution and observed in all kind of habitats of the world The
literature survey suggests that algae are capable of degrading azo dyes through
an induced form of an azoreductase (Jinqi and Houtian 1992) Color removal by
algae was due to three intrinsically different mechanisms of assimilative
utilization of chromophores for production of algal biomass CO2 and H2O
transformation of colored molecules to non-colored molecules and adsorption of
chromophores on algal biomass Several species of Chlorella (Acuner and Dilek
2004) and Oscillatoria (Jinqi and Houtian 1992) were capable of degrading azo
dyes to their aromatic amines and to further metabolize the aromatic amines to
simpler organic compounds or CO2 Mohan et al (2002) attributes the
decolorization to biosorption followed by bioconversion and biocoagulation It
was reported that more than 30 azo compounds were biodegraded and
decolorized by Chlorella pyrenoidosa Chlorella vulgaris and Oscillateria
tenuis in which azo dyes were decomposed into simpler aromatic amines (Yan
and Pan 2004) Thus the foregoing results could mean that algae can play an
20
Review of literature
important role in the removal of azo dyes and aromatic amines in stabilization
ponds (Banat et al 1996)
216 Decolorization with plant (phytoremediation)
Phytoremediation is considered as a plausible approach for the
remediation of soils and groundwater contaminated with heavy metals and
organic pollutants Recently some studies describe the use of plants for the dye
removal from wastewaters The Rheum rabarbarum mentions a good removal
capacity of sulfonated anthraquinones (Aubert and Schwitzgueacutebel 2004)
Recently narrow-leaved cattails were studied in synthetic reactive dye
wastewater treatment under caustic conditions (Nilratnisakorn et al 2007) Also
Mbuligwe describes a reduction in color of 72-77 in wetlands vegetated with
coco yam plants (Mbuligwe 2005) It was reported that the plant possesses
enzymes that accept anthraquinones as substrates and in cell culture were able to
remove up to 700-800 mg l-1 of anthraquinones with sulphonate groups in
different positions The three plant species (Brassica juncea Sorghum vulgare
and Phaseolus mungo) of different agronomic consequence were evaluated for
the decolorization of the dyes from textile effluent These plants B juncea S
vulgare and P mungo showed textile effluent decolorization up to 79 57 and
53 respectively (Ghodake et al 2008) Similarly an herb Blumea malcommi
was found to degrade textile dye Reactive Red 5B (Kagalkar et al 2009)
However in large scale application of phytoremediation presently faces a
number of obstacles including the level of pollutants tolerated by the plant the
bioavailable fraction of the contaminants and evapotranspiration of volatile
organic pollutants as well as requiring big areas to implant the treatment
(Williams 2002)
217 Bacterial decolorization and degradation of azo dyes
21
Review of literature
Generally the decolorization of azo dyes occurs under conventional
anaerobic anoxic and aerobic conditions by different groups of the bacteria The
mechanism of microbial degradation of azo dyes involves the reductive cleavage
of azo bonds (-N=N-) with the help of azoreductase under anaerobic conditions
resulted into the formation of colorless solutions containing potentially
hazardous-aromatic amines (Chang et al 2000 van der Zee and Villaverde
2005) The resulting intermediate metabolites (eg aromatic amines) are further
degraded aerobically or anaerobically (Seshadri et al 1994) Many recent
researches focus on utilization of microbial biocatalyst to reduce the dye from
the effluent (Hu 1998 Chang et al 2000 Martina Mohorčič 2004) Moreover
the effectiveness of microbial decolorization depends on the adaptability and the
activity of selected microorganisms It has been reported that a wide range of
microorganisms including bacteria fungi yeasts actinomycetes and algae are
capable of degrading azo dyes Moreover most studies on azo dye
biodegradation have focused on the bacteria and fungi The fungal cultures
mainly belonging to white rot fungi have been used to develop bioprocesses for
the mineralization of azo dyes (Parshetti et al 2007) However a long growth
cycle requiring nitrogen limiting conditions and long hydraulic retention time
for complete decolorization still limit the performance of the fungal
decolorization system (Banat et al 1996 Chang et al 2004 Jadhav et al
2008) In contrast bacterial decolorization and degradation of azo dyes has been
of considerable interest since it possesses higher degree of biodegradation and
mineralization diversity towards variety of azo dyes inexpensive and
eco-friendly nature and less sludge producing properties (Verma and Madmawar
2003 Rai et al 2005 Saratale et al 2009c Kalyani et al 2008 Dhanve et al
2008 Saratale et al 2006) Extensive studies have been carried out to determine
22
Review of literature
the role of the diverse groups of bacteria in the decolorization of azo dyes
(Pandey et al 2007)
i) Using pure bacterial culture
The effluents from textile industries are complex containing a wide
variety of dyes and other products such as dispersants acids bases salts
detergents humectants oxidants etc Discharge of these colored effluents into
rivers and lakes results into reduced dissolved oxygen concentration thus
creating anoxic conditions that are lethal to resident organisms Biological
processes provide an alternative to existing technologies because they are more
cost-effective environmentally friendly and do not produce large quantities of
sludge Bacterial decolorization is normally faster compared to fungal system for
the decolorization and mineralization of azo dyes It was observed that the mixed
cultures are apparent as some microbial consortia can collectively carry out
biodegradation tasks that no individual pure strain can undertake successfully
(Nigam et al 1996) However mixed cultures only provide an average
macroscopic view of what is happening in the system and results are not easily
reproduced making thorough effective interpretation difficult For these
reasons a substantial amount of research on the subject of color removal has been
carried out using single bacterial cultures like P mirabilis P luteola
Pseudomonas sp Pseudomonas sp SUK1 and K rosea has shown very
promising results for the dye degradation under anoxic conditions (Chen et al
1999 Chang et al 2001 Yu et al 2001 Kalyani et al 2008 Parshetti et al
2006) In addition there are also several studies describing the decolorization of
reactive dyes mediated by pure bacterial culture such as Exiguobacterium sp
RD3 for Navy Blue HE2R (Reactive Blue 172) (Dhanve et al 2008) Rhizobium
radiobacter MTCC 8161 for Reactive Red 141 (Telke et al 2008) P vulgaris
23
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
important role in the removal of azo dyes and aromatic amines in stabilization
ponds (Banat et al 1996)
216 Decolorization with plant (phytoremediation)
Phytoremediation is considered as a plausible approach for the
remediation of soils and groundwater contaminated with heavy metals and
organic pollutants Recently some studies describe the use of plants for the dye
removal from wastewaters The Rheum rabarbarum mentions a good removal
capacity of sulfonated anthraquinones (Aubert and Schwitzgueacutebel 2004)
Recently narrow-leaved cattails were studied in synthetic reactive dye
wastewater treatment under caustic conditions (Nilratnisakorn et al 2007) Also
Mbuligwe describes a reduction in color of 72-77 in wetlands vegetated with
coco yam plants (Mbuligwe 2005) It was reported that the plant possesses
enzymes that accept anthraquinones as substrates and in cell culture were able to
remove up to 700-800 mg l-1 of anthraquinones with sulphonate groups in
different positions The three plant species (Brassica juncea Sorghum vulgare
and Phaseolus mungo) of different agronomic consequence were evaluated for
the decolorization of the dyes from textile effluent These plants B juncea S
vulgare and P mungo showed textile effluent decolorization up to 79 57 and
53 respectively (Ghodake et al 2008) Similarly an herb Blumea malcommi
was found to degrade textile dye Reactive Red 5B (Kagalkar et al 2009)
However in large scale application of phytoremediation presently faces a
number of obstacles including the level of pollutants tolerated by the plant the
bioavailable fraction of the contaminants and evapotranspiration of volatile
organic pollutants as well as requiring big areas to implant the treatment
(Williams 2002)
217 Bacterial decolorization and degradation of azo dyes
21
Review of literature
Generally the decolorization of azo dyes occurs under conventional
anaerobic anoxic and aerobic conditions by different groups of the bacteria The
mechanism of microbial degradation of azo dyes involves the reductive cleavage
of azo bonds (-N=N-) with the help of azoreductase under anaerobic conditions
resulted into the formation of colorless solutions containing potentially
hazardous-aromatic amines (Chang et al 2000 van der Zee and Villaverde
2005) The resulting intermediate metabolites (eg aromatic amines) are further
degraded aerobically or anaerobically (Seshadri et al 1994) Many recent
researches focus on utilization of microbial biocatalyst to reduce the dye from
the effluent (Hu 1998 Chang et al 2000 Martina Mohorčič 2004) Moreover
the effectiveness of microbial decolorization depends on the adaptability and the
activity of selected microorganisms It has been reported that a wide range of
microorganisms including bacteria fungi yeasts actinomycetes and algae are
capable of degrading azo dyes Moreover most studies on azo dye
biodegradation have focused on the bacteria and fungi The fungal cultures
mainly belonging to white rot fungi have been used to develop bioprocesses for
the mineralization of azo dyes (Parshetti et al 2007) However a long growth
cycle requiring nitrogen limiting conditions and long hydraulic retention time
for complete decolorization still limit the performance of the fungal
decolorization system (Banat et al 1996 Chang et al 2004 Jadhav et al
2008) In contrast bacterial decolorization and degradation of azo dyes has been
of considerable interest since it possesses higher degree of biodegradation and
mineralization diversity towards variety of azo dyes inexpensive and
eco-friendly nature and less sludge producing properties (Verma and Madmawar
2003 Rai et al 2005 Saratale et al 2009c Kalyani et al 2008 Dhanve et al
2008 Saratale et al 2006) Extensive studies have been carried out to determine
22
Review of literature
the role of the diverse groups of bacteria in the decolorization of azo dyes
(Pandey et al 2007)
i) Using pure bacterial culture
The effluents from textile industries are complex containing a wide
variety of dyes and other products such as dispersants acids bases salts
detergents humectants oxidants etc Discharge of these colored effluents into
rivers and lakes results into reduced dissolved oxygen concentration thus
creating anoxic conditions that are lethal to resident organisms Biological
processes provide an alternative to existing technologies because they are more
cost-effective environmentally friendly and do not produce large quantities of
sludge Bacterial decolorization is normally faster compared to fungal system for
the decolorization and mineralization of azo dyes It was observed that the mixed
cultures are apparent as some microbial consortia can collectively carry out
biodegradation tasks that no individual pure strain can undertake successfully
(Nigam et al 1996) However mixed cultures only provide an average
macroscopic view of what is happening in the system and results are not easily
reproduced making thorough effective interpretation difficult For these
reasons a substantial amount of research on the subject of color removal has been
carried out using single bacterial cultures like P mirabilis P luteola
Pseudomonas sp Pseudomonas sp SUK1 and K rosea has shown very
promising results for the dye degradation under anoxic conditions (Chen et al
1999 Chang et al 2001 Yu et al 2001 Kalyani et al 2008 Parshetti et al
2006) In addition there are also several studies describing the decolorization of
reactive dyes mediated by pure bacterial culture such as Exiguobacterium sp
RD3 for Navy Blue HE2R (Reactive Blue 172) (Dhanve et al 2008) Rhizobium
radiobacter MTCC 8161 for Reactive Red 141 (Telke et al 2008) P vulgaris
23
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
Generally the decolorization of azo dyes occurs under conventional
anaerobic anoxic and aerobic conditions by different groups of the bacteria The
mechanism of microbial degradation of azo dyes involves the reductive cleavage
of azo bonds (-N=N-) with the help of azoreductase under anaerobic conditions
resulted into the formation of colorless solutions containing potentially
hazardous-aromatic amines (Chang et al 2000 van der Zee and Villaverde
2005) The resulting intermediate metabolites (eg aromatic amines) are further
degraded aerobically or anaerobically (Seshadri et al 1994) Many recent
researches focus on utilization of microbial biocatalyst to reduce the dye from
the effluent (Hu 1998 Chang et al 2000 Martina Mohorčič 2004) Moreover
the effectiveness of microbial decolorization depends on the adaptability and the
activity of selected microorganisms It has been reported that a wide range of
microorganisms including bacteria fungi yeasts actinomycetes and algae are
capable of degrading azo dyes Moreover most studies on azo dye
biodegradation have focused on the bacteria and fungi The fungal cultures
mainly belonging to white rot fungi have been used to develop bioprocesses for
the mineralization of azo dyes (Parshetti et al 2007) However a long growth
cycle requiring nitrogen limiting conditions and long hydraulic retention time
for complete decolorization still limit the performance of the fungal
decolorization system (Banat et al 1996 Chang et al 2004 Jadhav et al
2008) In contrast bacterial decolorization and degradation of azo dyes has been
of considerable interest since it possesses higher degree of biodegradation and
mineralization diversity towards variety of azo dyes inexpensive and
eco-friendly nature and less sludge producing properties (Verma and Madmawar
2003 Rai et al 2005 Saratale et al 2009c Kalyani et al 2008 Dhanve et al
2008 Saratale et al 2006) Extensive studies have been carried out to determine
22
Review of literature
the role of the diverse groups of bacteria in the decolorization of azo dyes
(Pandey et al 2007)
i) Using pure bacterial culture
The effluents from textile industries are complex containing a wide
variety of dyes and other products such as dispersants acids bases salts
detergents humectants oxidants etc Discharge of these colored effluents into
rivers and lakes results into reduced dissolved oxygen concentration thus
creating anoxic conditions that are lethal to resident organisms Biological
processes provide an alternative to existing technologies because they are more
cost-effective environmentally friendly and do not produce large quantities of
sludge Bacterial decolorization is normally faster compared to fungal system for
the decolorization and mineralization of azo dyes It was observed that the mixed
cultures are apparent as some microbial consortia can collectively carry out
biodegradation tasks that no individual pure strain can undertake successfully
(Nigam et al 1996) However mixed cultures only provide an average
macroscopic view of what is happening in the system and results are not easily
reproduced making thorough effective interpretation difficult For these
reasons a substantial amount of research on the subject of color removal has been
carried out using single bacterial cultures like P mirabilis P luteola
Pseudomonas sp Pseudomonas sp SUK1 and K rosea has shown very
promising results for the dye degradation under anoxic conditions (Chen et al
1999 Chang et al 2001 Yu et al 2001 Kalyani et al 2008 Parshetti et al
2006) In addition there are also several studies describing the decolorization of
reactive dyes mediated by pure bacterial culture such as Exiguobacterium sp
RD3 for Navy Blue HE2R (Reactive Blue 172) (Dhanve et al 2008) Rhizobium
radiobacter MTCC 8161 for Reactive Red 141 (Telke et al 2008) P vulgaris
23
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
the role of the diverse groups of bacteria in the decolorization of azo dyes
(Pandey et al 2007)
i) Using pure bacterial culture
The effluents from textile industries are complex containing a wide
variety of dyes and other products such as dispersants acids bases salts
detergents humectants oxidants etc Discharge of these colored effluents into
rivers and lakes results into reduced dissolved oxygen concentration thus
creating anoxic conditions that are lethal to resident organisms Biological
processes provide an alternative to existing technologies because they are more
cost-effective environmentally friendly and do not produce large quantities of
sludge Bacterial decolorization is normally faster compared to fungal system for
the decolorization and mineralization of azo dyes It was observed that the mixed
cultures are apparent as some microbial consortia can collectively carry out
biodegradation tasks that no individual pure strain can undertake successfully
(Nigam et al 1996) However mixed cultures only provide an average
macroscopic view of what is happening in the system and results are not easily
reproduced making thorough effective interpretation difficult For these
reasons a substantial amount of research on the subject of color removal has been
carried out using single bacterial cultures like P mirabilis P luteola
Pseudomonas sp Pseudomonas sp SUK1 and K rosea has shown very
promising results for the dye degradation under anoxic conditions (Chen et al
1999 Chang et al 2001 Yu et al 2001 Kalyani et al 2008 Parshetti et al
2006) In addition there are also several studies describing the decolorization of
reactive dyes mediated by pure bacterial culture such as Exiguobacterium sp
RD3 for Navy Blue HE2R (Reactive Blue 172) (Dhanve et al 2008) Rhizobium
radiobacter MTCC 8161 for Reactive Red 141 (Telke et al 2008) P vulgaris
23
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
for Scarlet RR Navy Blue HE2R M glutamicus for Scarlet RR Reactive Green
19A (Saratale et al 2009b and c) and isolated bacterium KMK48 for the
degradation of various sulfonated reactive azo dyes (Kodam et al 2005) The
use of a pure culture system ensures the reproducible data and that the
interpretation of experimental observations is easier The detailed mechanisms of
biodegradation can be determined using the tools of biochemistry and molecular
biology These tools may also be used to up regulate the enzyme system to give
modified strains with enhanced activities In our laboratory the metabolic
pathway of particular dyes using pure culture was determined using various
analytical techniques such as Uv-Visible spectroscopy FTIR GCMS AAS
NMR etc The quantitative analysis of the kinetics of azo-dye decolorization by a
particular bacterial culture can be undertaken meaningfully Also the effects of
various physicochemical parameters on the pure bacterial culture were studied in
detail and some of the studies summaries in Table 21
ii) Using co-culture and mixed bacterial cultures
Bacterial decolorization was found more efficient and faster but
individual bacterial strain usually cannot degrade azo dyes completely The
intermediate products are often carcinogenic aromatic amines which need to be
further decomposed (Joshi et al 2008) Thus the treatment systems composed
of mixed microbial populations possess higher degree of biodegradation and
mineralization due to synergistic metabolic activities of microbial community
and offers considerable advantages over the use of pure cultures in the
degradation of synthetic dyes (Khehra et al 2005a) In the microbial consortium
the individual strains may attack the dye molecule at different positions or may
utilize metabolites produced by the co-existing strains for further decomposition
(Chang et al 2004 Forgacs et al 2004 Saratale et al 2009b Patil et al 2008)
24
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
25
Sphingomonas strain BN6 is able to degrade naphthalene-2-sulphonate a
building block of azo dyes into salicylate ion equivalents The salicylate ion
cannot be further degraded and it is toxic to strain BN6 Therefore
naphthalene-2-sulphonate can only be degraded completely in the presence of a
complementary organism that is capable of degrading the salicylate ion (Moosvi
et al 2005) In addition it can be difficult to isolate a single bacterial strain from
dye-containing wastewater samples and in some instances long term adaptation
procedures are necessary before the isolate is capable of using the azo dye as a
respiratory substrate Several studies reported in the literature concerning the
biodegradation of colored wastewater using mixed bacterial cultures are given in
Table 21
22 Mechanism of color removal
Azo compounds are susceptible to biological degradation under both
aerobic and anaerobic conditions (Field et al 1995) Decolorization of azo dyes
under anaerobic conditions is thought to be a relatively simple and non-specific
process involving fission of the azo bond to yield degradation products such as
aromatic amines The efficacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been many times
demonstrated (Delee et al 1998) Under anaerobic conditions a low redox
potential (lt50 mV) can be achieved which is necessary for the effective
decolorization of the dyes (Beydilli et al 1998 Bromley-Challenor et al 2000)
Color removal under anaerobic conditions is also referred as dye reduction in
which literature mostly covers the biochemistry of azo dye reduction
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
Table 21 Decolorization of various azo dyes by pure coculture consisting of pure bacterial culture and mixed bacterial culture
Name of strain Name of Dye and Concentration
Condition (pH temp (oC) agitation)
Time (h) Decolorization ()
Reaction Mechanism References
Pure bacterial culture Micrococcus glutamicus NCIM 2168
Reactive Green 19 A (50 mg1-1)
68 37 static
42 100 Oxidative and reductive Saratale et al 2009c
Rhizobium radiobacter MTCC 8161
Reactive red 141 (50 mg1-1)
70 30 static 48 90 Oxidative and reductive Telke et al 2008
Pseudomonas sp SUK1
Red BLI (50 mg1-1) 65 -70 30 static 1 99 AND and NADH-DCIP reduction
Kalyani et al 2007
Pseudomonas sp SUK1
Reactive Red2 (5 g1-1) 62-75 30 static 6 96 LiP and azoreductase Kalyani et al 2008
Pseudomonas desmolyticum NCIM 2112
Red HE7B (100 mg1-1) 68-80 30 static 72 95 LiP and azoreductase Kalme et al 2007
Comamonas sp UVS Direct red 5B (1100 mg1-1)
65 40 static 13 100 LiP and Laccase Jadhav et al 2008
Unidentified bacterium KMK 48
Reactive Red 2 Reactive Red 141 Reactive Orange 4
Neutral pH room temp aerobic
30 100 Reduction Kodam et al 2005
26
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
Reactive Orange 7 Reactive Violet 5 (200-1000 mg1-1 each)
Proteus mirabilis RED RBN (1g1-1)
65ndash75 30ndash35 static 20 95 Reductive followed by biosorption
Chen et al 1999
Exiguobacterium spRD3
Navy Blue HE2R (50 mg1-1)
70 30 static 48 91 LiP Laccase and azoreductase
Dhanve et al 2008
Aeromonas hydrophila
Red RBN (3000 mg1-1)
55-100 20-35 NA 8 90 NA Chen et al 2003
Pseudomonas aeruginosa NBAR12
Reactive Blue 172 (500 mg1-1)
70-80 40 static 42 83 Azoreductases flavin reductases or peroxidases
Bhatt et al 2005
Bacillus sp Congo Red (100ndash300 mg1-1)
70 37 NA 24ndash27a 12b
100a
100b
Effect of sonication Kannappan et al 2009
K1ebsi1e11a pneumoniae R5-13
Methy1 Red (100 mg1-1)
60-80 30 200 rpm 168 100 Reduction Wong and Yuen 1996
Rhodopseudomonas palustris AS12352
Reactive Brilliant Red X-3B (50 mg1-1)
8 30ndash35 anaerobic 24 90 Azoreduction Liu et al 2006
Citrobacter sp Azo and Triphenylmethane dyes (5 microM)
7ndash9 35ndash40 static 1 100 Adsorption An et al 2002
S chromofuscus A11 Various azo dyes (50 mg1-1 each)
NA 37 200 rpm 168 70-98 NA Pasti-Grisby et al 1996
27
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
Pseudomonas luteola RP2B (Reactive Red 22) (100 ppm)
NA 28 Shaking-static incubation (100 rpm for 48 h then at static for 4 days)
120 95 Azoreduction Hu 1998
Shewanella putrefaciens AS96
Acid Red 88 Direct Red81 Reactive Black 5 Dieperse Orange 3 (mixture of these 4 dyes) (100 mg1-1)
NA NA Static
4 6 8
100 100 100
NA Khalid et al 2008
Shewanella decolorationis S12
Acid Red GR NA 30 c and d 68c
10d
100c
100d
Azoreduction Xu et al 2007
Paenibacillus azoreducens sp nov
Remazol Black B NA 37 100 mg dm3
24 98 NA Meehan et al2001
Shewanella putrefaciens
Remazol Black B 8 reactive and anthraquinone dye
8 35 static NA 95 NA Bragger et al1997
Desulfovibrio desulfuricans
CI Reactive Orange 96 NA 28 Anaerobic 2 95 Reduction Yoo et al 2000
Pseudomonas luteola
Reactive azo dyes Direct azo dyes and leather
NA Static NA 24-144 59ndash99 NA Hu 2001
28
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
dyes (100 mg1-1)
Bacillus fusiformis KMK5
Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) (15 g1-1)
NA anoxic NA 48 100 Azoreductase Kolekar et al 2008
Aeromonas hydrophila var 24 B
Various azo dyes (10-100 mg1-1)
NA 24 50-90
Azoreductase (cell free extract)
Idaka and Ogawa 1978
Bacillus subtilis IFO 13719
2-carboxy 4- dimethyleamino benzene (0045 mmol)
NA 20 min
100 Azoreductase (in growing cells)
Yatome et al 1991
Klebsiella pneumoniae RS-13
Methyl Red (100 mg1-1)
NA 24 100
Azoreduction Wong and Yuen 1996
Sphingomonas sp BN6
Acid azo dyes Direct azo dyes and Amaranth
NA NA Reduction Russ et al 2000
Pseudomonas luteola
CI Reactive Red 22 Agitation without aeration and with a constant dye loading rate of 200 mg h-1
NA 1137 mg dye g cell-1 h-1
NA Chang and Lin 2000
Sphingomonas sp BN6
Amaranth (sulfonated azo dye)
NA NA Azoreduction Keck and Kudlich et al 1997
Bacillus subtilis p-Aminoazobenzene (PAAB)
NA anoxic NA 25 100 NA Zissi and Lyberatos 1996
29
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
(102 to 75 mg1-1)
Pseudomonas luteola
4 reactive azo dyes NA static NA 42 37ndash93 NA Hu 1990
Pseudomonas cepacia 13NA
CI Acid Orange 12 CI Acid Orange 20 CI Acid Red 88
NA 68 90 NA Ogawa et al 1986
Pseudomonas sp Orange I Orange II
NA 35 90 Azoreduction Kulla et al 1983
Trichosporon beigelii NCIM-3326
Navy Blue HER (50 mg1-1)
70 37 static
24
100
Oxidative and reductive Saratale et al 2009b
Desulfovibrio desulfuricans
Reactive Orange 96 Reactive Red 120 (03 mmol L-1)
113 37 NA Within 2 h from 360 to 362 h
95 Reduction Yoo et al 2000
Micrococcus glutamicus NCIM 2168
Scarlet RR and mixture of 12 dyes (50 mg1-1 each)
69 37 static 20 100 Oxidative and reductive This study
Proteus vulgaris NCIM 2027
Green HE4BD (50 mg1-1)
70 37 static
72
100
Reduction This study
Proteus vulgaris NCIM 2027
Scarlet RR Navy Blue HE2R (50 mg1-1each)
70 37 static 70 37 static
14 9
100 100
Reduction This study
Co culture consisting of pure bacterial culture
30
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
Consortium-GR (P vulgaris and M glutamicus)
Scarlet RR and mixture of 8 dyes (50 mg1-1 each)
70 37 static
3 100 Reduction
Saratale et al 2009a
Consortium-GR (P vulgaris and M glutamicus)
Green HE4BD and mixture of 10 dyes (50 mg1-1)
68 37 static
24 100 Oxidative and reductive This study
Enterobacter sp Serratia sp Yersinia sp Erwinia sp
Reactive Red 195 (30 mg1-1)
70 37 150 rpm 48 90 NA Jirasripongpun et al 2007
Consortium two isolated strains (BF1 BF2) and a strain of Pseudomonas putida (MTCC-1194)
Direct Yellow 86 Basic Orange 2 Reactive Green 19 Direct Blue 54 Reactive Blue 171 Reactive Red 141 Acid Red 260
(50 mg1-1 each)
9ndash105 28 120 rpm 168 80 NA Resmi et al 2004
Microbial consortium(white-rot fungus 8-4 amp Pseudomonas 1-10)
Direct Fast Scarlet 4BS (50 mg1-1)
4-9 20-40 NA 24 991 NA Fang et al 2004
Four bacterial isolates consortium Bacillus cereus (BN-7)
Acid Red 88 Acid Red 119 Acid Red 97
70 35 100 rpm 24 24 24
78 99 94
Azoreduction Khehra et al 2005
31
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
Pseudomonas putida (BN-4) Pseudomonas fluorescence (BN-5) and Stenotrophomonas acidaminiphila (BN-3)
Acid Blue 113 Reactive Red 120 (60 mg1-1 each)
24 24
99 82
Mixed bacterial consortium JW-2
Reactive Violet 5R (100 ppm)
65 to 85 25 to 37 static 36 100 NA Moosvi et al 2007
Bacterial consortium (TJ-1) Aeromonas
caviae Proteus mirabilis and
Rhodococcus globerulus
Acid Orange 7 (AO7) (200 mg1-1)
NA 30 microaerophilic condition
16 90 NA Joshi et al 2008
Bacterial consortium SV5
Ranocid Fast Blue (100 ppm)
70 37 static 24 100 NA Mathew and Madamwar 2004
Bacterial consortium Direct Red 81 (DR 81) (200 mg1-1)
70 37 NA 35 90 NA Junnarkar et al 2006
Cyanobacterial cultures isolated (Gloeocapsa pleurocapsoides and Chroococcus minutus)
Acid Red 97 and FF Sky Blue Amido Black 10B (100 mg1-1)
NA 27 static 26 days 78-90 Oxidative enzymes Parikh and Madamwar 2005
Acclimatized Reactive Black 5 NA 37 static anaerobic 48 70ndash90 Enzyme secretion Dafale et al 2007
32
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
Microbial consortium
Pseudomonas aeruginosa and Bacillus circulans and (NAD1 amp NAD6) isolates
(100 mg1-1) batch
Five-member bacterial consortium
Alcaligenes faecalis Sphingomonas sp EBD Bacillus subtilis Bacillus thuringiensis and Enterobacter
cancerogenus
Direct Blue-15 (50 mg1-1)
NA 37 static 24 9214 NA Kumar et al 2007
Bacterial consortium RVM111
Reactive Violet 5 (200 mg1-1)
65 to 85 25 to 40 NA 37 94 NA Moosvi et al 2005
Consortium of
Alcaligenes faecalis Commomonas acidovorans
Remazol Black B NA 48 95 NA Oxspring et al 1996
Mixed bacterial cultureMixed culture Reactive Black 5 60-70 RT NA 48 90 Two stage anaerobic- Mohanty et al
33
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
(100-3000 mg1-1) aerobic treatment 2006 Dried activated sludge Reactive Black 5
(116 mg1-1) 20 20 NA 30 min 50 Adsorption Gulnaz 2005
Mixed bacterial cultures
Mixture of azo- and diazo-reactive dyes
NA 96 80 NA Nigam et al 1996
Mixed bacterial cultures
Diazo-linked chromophore
Use of triple mix gas H2-10 CO2-10 N2-80 at 30degC strictly anaerobic conditions
48 85 Flavin coenzymes
Knapp et al (1995)
Mixed cultures Reactive and disperse textile dyes (05 g1-1)
6 37 120 rpm 120-240 100 NA Asgher et al 2007
Methanogenic and Mixed bacteria cultures
CI Acid Orange 7 (60-300 mg1-1)
NA 37 NA 140 96 The anaerobic batch reactors
Braacutes et al 2001
Isolated halophilic and halotolerant bacteria
Textile azo dyes including Remazol Black B Maxilon Blue Sulphonyl Scarlet BNLE Sulphonyl Blue TLE Sulphonyl Green BLE Remazol Black N and Entrazol Blue IBC (5000 ppm)
5ndash11 25ndash40 static 96 100 Azoreductase Asad et al 2007
34
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
Mixed culture of bacteria
Remazol Brilliant Orange 3R Remazol Black B and Remazol Brilliant Violet 5R (05-10 g1-1)
NA 30 200 rpm sequential anaerobic-aerobic treatment process
24 24
789 90
NA
Supaka 2004
Mixed-culture Methyl Red 6 30 NA 16 82 Azoreduction Adedayo et al 2004
Mixed microbial culture
Direct Black-38 (100 mg1-1)
NA 37 static 240 100 NA Kumar et al 2006
Activated sludge obtained from domestic and industrial
effluent treatment plants
Reactive Red 31 (20-30 mg 1-1)
Anaerobic packed bed reactor followed by an aerobic stirred tank reactor
51 90ndash93 NA Bromley-Challenor et al 2004
Original seed sludge collected from a municipal wastewater treatment plant
CI Acid Red 42 CI Acid Red 73 CI Direct Red 80 CI Disperse Blue 56
NA 80 90 NA Goncalves et al 2000
Mixed bacterial population with sulphate-reducing bacteria and a methanogenic
Wastewater containing
up to 15 different sulfonated azo dyes
An anaerobic baffled reactor
NA NA NA Plumb et al 2001
35
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
population Mixed and methanogenic cultures
CI Acid Orange 7 NA NA 94 Azoreduction Bras et al 2001
Sulphate-reducing bacteria methane producing bacteria and fermentative bacteria in an anaerobic mixed culture
Hydrolysed CI Reactive Orange 96
NA 40 30
95 90
NA Yoo et al 2001
An uncharacterized aerobic biofilm Strain 1CX (Sphinogomonas sp) and Strain SAD4I (Gram-negative bacterium)
Acid Orange 7 A rotating drum bioreactor containing the biofilm
1 90 NA Coughlin et al 2002
Bacillus cereus Sphaerotilus natans Arthrobacter sp activated sludge
Azo dyes NA NA Anoxic conditions NA NA Reduction Wuhrmann et al 1980
36
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
Field-collected and laboratory cultures
CI Acid Orange 6 CI Basic Violet 1 CI Basic Violet 3
NA NA Aerobic condition NA NA NA Michaels and Lewis 1986
Anaerobic digester sludge and aeration tank mixed liquor
CI Acid Orange 10 CI Acid Red 14 CI Acid Red 18
2-stage anaerobic-aerobic (fixed film fluidised bedsuspended growth activated sludge) reactor
NA 65ndash90 NA Fitzgerald and Bishop 1995
Alcaligenes faecalis Commomonas acidovorans
Reactive dyes diazo dyes azo dyes disperse dyes and phthalocyanine dyes
Anaerobic conditions 48 100 NA Nigam et al 1996
Aerobic mixed bacterial culture
Cationic chromium- containing azomethine dye
NA NA NA Adsorption Matanic et al 1996
Thermophilic anaerobic bacterial culture
Various azo and diazo reactive dyes
NA 48 68ndash84 NA Banat et al 1996
Sludge from textile wastewater treatment plant (Inclined Tubular Digester) granules
Simulated textile wastewater containing Procion Red HE7B
Upflow Anaerobic Sludge Blanket (UASB)
NA 78 NA OrsquoNeil et al 1999
37
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
38
from paper pulp processing plant (Upflow Anaerobic Sludge Blanket)
Four bacterial strains (Pseudomonas) isolated from dyeing
effluent-contaminated soils
Orange G Amido Black 10B Direct Red 4BS Congo Red
NA 1125 1349 mg l-1 d-1
609
NA Rajaguru et al 2000
Mixed culture from upward-flow anaerobic sludge bed (UASB) reactor
20 selected azo dyes (100-300 mglminus1)
NA 1 to 100 100 NA van der Zee et al 2001
a Without sonication pretreatment b With sonication pretreatment c Anaerobic condition d Microaerophilic DO range 02 to 05 mgl-1 150 rpm NA- Not available
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
The mechanism of microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (ndashN=Nndash) with the help of azoreductase under anaerobic
conditions involves a transfer of four-electrons (reducing equivalents) which
proceeds through two stages at the azo linkage and in each stage two electrons
are transferred to the azo dye which acts as a final electron acceptor resulted
into dye decolorization and the formation of colorless solutions The resulting
intermediate metabolites (eg aromatic amines) are further degraded aerobically
or anaerobically (Chang et al 2000 2004) Thus in the presence of oxygen
usually inhibits the azo bond reduction activity since aerobic respiration may
dominate utilization of NADH thus impeding the electron transfer from NADH
to azo bonds (Chang et al 2004) The potential toxicity mutagenicity and
carcinogenicity of such compounds are well documented and have been
reviewed elsewhere (Chung et al 1992)
Reaction mechanism
(R1ndashN=NndashR2 ) + 2e_ +2H+ mdashgt R1ndashHNndashNHndashR2
(Hydrazo intermediate)
(R1ndashHNndashNHndashR2)+ 2e_ +2H+ mdashgt R1-NH2+ R2-NH2
(Reductive cleavage of the azo bond)
Much of the experimental work involving the anaerobic decolorization of dyes
(predominantly azo dyes) was conducted using mono cultures Species of
Bacillus Pseudomonas Aeromonas Proteus Micrococcus and purple
non-sulphur photosynthetic bacteria were found to be effective in the anaerobic
degradation of a number of dyes (Horitsu et al 1977 Chang et al 2001
Saratale et al 2009bc Kalme et al 2008) The anaerobic reduction of azo dyes
39
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
utilized in the textile industry by a strain of Bacillus cereus isolated from soil
(Wuhrmann et al 1980) However the permeation of the dyes through
biological membrane into the microbial cells was cited as the principal
rate-limiting factor for decolorization (Kodam et al 2006)
In contrast under aerobic conditions the enzymes mono- and
di-oxygenase catalyze the incorporation of oxygen from O2 into the aromatic
ring of organic compounds prior to ring fission (Madigan et al 2003) However
in the presence of specific oxygen-catalysed enzymes called azo reductases
some aerobic bacteria are able to reduce azo compounds and produce aromatic
amines (Stolz 2001) Examples of aerobic azo reductases were found in
Pseudomonas species strains K22 and KF46 (Zimmermann et al 1982 1984)
These enzymes after purification characterization and comparison were shown
to be flavin-free The aerobic azo reductases were able to use both NAD(P)H and
NADH as cofactors and reductively cleaved not only the carboxylated growth
substrates of the bacteria but also the sulfonated structural analogues Recently
Blumel and Stolz (2003) cloned and characterized the genetic code of the aerobic
azo reductase from Pagmentiphaga kullae K24 Only few bacteria with
specialized azo dye reducing enzymes were found to degrade azo dyes under
fully aerobic conditions (Zimmerman et al 1882 1984 Nachiyar et al 2003
Nachiyar et al 2005)
23 Factors affecting bacterial decolorization
Optimization of operating system is necessary to obtain maximum rate of
decolorization of azo dyes The efficiency of biological treatment systems is
greatly influenced by the various physicochemical parameters such as level of
aeration temperature pH the dye structure its initial concentration effect of
different media composition and supplementation of different carbon and
40
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
nitrogen sources to enhance the decolorization performance The concentrations
of the electron donor and the redox mediator must be optimized with the amount
of biomass in the system and the quantity of dye present in the wastewater to
improve the further treatment process The chemical composition of textile
wastewater is usually subject to daily and seasonal variations and includes
organics nutrients sulphur compounds salts and different toxic substances
(Pearce et al 2003) Presence of these compounds may have an inhibitory effect
on the dye decolorization process The ability of the bacterial cells for dye
decolorization must be determined at different conditions since the dyes are
intentionally designed to resist degradation and their concentration may have
low removal efficiency by various treatments systems (Philippe et al 1998)
Therefore the effect of each of the factors on the dye decolorization must be
determined prior to the treatment of the industrial wastewater by biological
means
231 Oxygen
The most important factor to consider is the effect of oxygen on the cell
growth and dye reduction It was observed that during the cell growth stage
oxygen will have a significant effect on the physiological characteristics of the
cells During the azo dye reduction cleavage stage with the help of azoreductase
if the extra cellular environment is aerobic the high-redox-potential electron
acceptor oxygen may inhibit the dye reduction mechanism (Chang et al 2004)
Similar results were observed in the studies on pure bacterial strains such as
Pseudomonas luteola Proteus mirabilis Pseudomonas sp SUK1 Proteus
vulgaris NCIM-2027 Micrococcus glutamicus NCIM-2168 (Chen et al 1999
Chang et al 2001 Kalyani et al 2008 Saratale et al 2009b Saratale et al
2009c) This is because of the electrons liberated from the oxidation of electron
41
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
donors by the cells are preferentially used to reduce oxygen rather than the azo
dye and the reduction product water is not a reductant (Yoo et al 2001) Also
the postulated intermediates of the dye reduction reaction which include the
hydrazine form of the dye and the azo anion free radical form of the dye tend to
reoxidize by molecular oxygen (Zimmerman et al 1982) From these
observations it was recommended that for efficient color removal aeration and
agitation which increases the concentration of oxygen in solution should be
avoided (Chang and Lin 2000) It was also noteworthy that inhibition of azo dye
reduction under aerobic conditions inclines only to be a temporary effect rather
than an irreversible effect If the air is replaced with oxygen free nitrogen the
reducing activity is restored and occurs at a similar rate to that which was
observed under continuous anaerobic conditions (Bragger et al 1997)
However aerobic conditions are required for the complete mineralization
of the reactive azo dye molecule as the simple aromatic compounds produced by
the initial reduction are degraded via hydroxylation and ring-opening in the
presence of oxygen (Chang et al 2001 Pandey et al 2007) Thus for the most
effective wastewater treatment a two-stage process is necessary in which oxygen
is introduced after the initial anaerobic reduction of the azo bond has taken place
The balance between the anaerobic and aerobic stages in this treatment system
must be carefully controlled because it is possible for the re-aeration of a reduced
dye solution to cause the color of the solutions to darken However when the
correct operating conditions have been established many strains of bacteria are
capable of achieving high levels of decolorization when used in a sequential
anaerobicaerobic treatment process (Khehra et al 2006)
42
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
232 Temperature
The literature survey suggests that the rate of color removal increases
with an increasing temperature up to certain limit afterwards there is marginal
reduction in the decolorization activity It was observed that the temperature
required to produce the maximum rate of color removal tends to correspond with
the optimum cell culture growth temperature of 35ndash45 degC The decline in color
removal activity at higher temperatures can be attributed to the loss of cell
viability or due to the denaturation of an azo reductase enzyme (Chang et al
2001 Telke et al 2008 Saratale et al 2009b) However it has been shown that
with certain whole bacterial cell preparations the azo reductase enzyme is
relatively thermo stable and can remain active up to temperatures of 60 degC over
short periods of time (Pearce et al 2003) Immobilization of the cell culture in a
support medium results in a shift in the optimum color removal temperature
towards high values because the microenvironment inside the support offers
protection for the cells (Chang et al 2001 Pearce et al 2003)
233 pH
The medium pH is also important factor for better decolorization activity
In many studies it was observed that the optimum pH for color removal is often
at a neutral pH or at slightly alkaline pH The rate of color removal was higher at
only optimum pH but tends to decrease rapidly at strongly acid or strongly
alkaline pH In our laboratories similar results were observed with many
microbial strains (Table 21) The interesting results were observed with the
consortium-GB consisting of Galactomyces geotrichum MTCC1360 and
Bacillus sp VUS where the decolorization was not pH dependent Complete
decolorization was observed in the pH range from 5 to 9 (Jadhav et al 2008)
Biological reduction of the azo bond can result in an increase in the pH due to the
43
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
formation of aromatic amine metabolites which are more basic then the original
azo compound (Willmott 1997) Altering the pH within a range of 70 to 95
has very little effect on the dye reduction process Chang et al (2001) found that
the dye reduction rate increased nearly 25-fold as the pH was raised from 50 to
70 while the rate became insensitive to pH in the range of 70ndash95
234 Dye concentration
The concentration of dye substrate can influence the efficiency of dye
removal through a combination of factors including the toxicity of the dye (and
co-contaminants) at higher concentrations and the ability of the enzyme to
recognize the substrate efficiently at very low concentrations that may be present
in some wastewaters Decrease in the decolorization rates may occur due to the
toxicity of the dye to bacterial cells andor inadequate biomass concentration (or
improper cell to dye ratio) for the uptake of higher concentrations of dye
(Saratale et al 2006 Jadhav et al 2008a) Similar results were observed by
isolated Exiguobacterium sp RD3 Pseudomonas sp SUK1 Proteus vulgaris
NCIM 2027 Micrococcus glutamicus NCIM-2168 for the decolorization of
various reactive group of azo dyes (Dhanve et al 2008 Kalyani et al 2008
Saratale et al 2009b Saratale et al 2009c) It was also observed that reactive
group azo dyes having sulfonic acid (SO3H) groups on aromatic rings greatly
inhibited the growth of microorganisms at higher dye concentration (Chen et al
2003 Kalyani et al 2008) Sani and Banerjee (1999) found that dyes with
concentrations of 1-10 μM were easily decolorized but when the dye
concentration was increased to 30 μM color removal was reduced Surprisingly
Dubin and Wright (1975) reported the absence of any effect of dye concentration
on the reduction rate This observation is compatible with a non-enzymatic
reduction mechanism that is controlled by processes that are independent of the
44
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
dye concentration Moreover the kinetic constants that govern process efficiency
in common with other enzyme catalysed processes can be described using
MichaelisndashMenten kinetics
where v is the observed velocity of the reaction at a given substrate concentration
[S] Vmax is the maximum velocity at a saturating concentration of substrate and
Km is the Michaelis constant The application of MichaelisndashMenten kinetics can
allow predictions to be made on process efficiency including the degree of
biomass loading or the operational temperature needed to maintain dye removal
at a given efficiency within the constraints set by the reactor volume available
the background solution composition and flow-rates
235 Dye structure
The chemical structure of textile dyes also affects the bacterial
decolorization performance It was reported that dyes with simple structures and
low molecular weights exhibit higher rates of color removal whereas color
removal rate is lower in the case of dye with substitution of electron withdrawing
groups viz (ndashSO3H ndashSO2NH2) in the para position of the phenyl ring relative to
the azo bond and high molecular weight dyes (Sani and Banerjee 1999) Nigam
et al (1996) established that azo compounds with a hydroxyl group or with an
amino group are more likely to be degraded than those with a methyl methoxy
sulpho or nitro groups Moreover color removal rate is also related to the number
of azo bonds in the dye molecule like for monoazo dyes the color removal rate is
faster than the diazo or triazo dyes (Hu 2001) Hitz et al (1978) depicts that
decolorization rate is dependent on dye class like (a) acid dyes exhibit low color
removal due to the number of sulphonate groups in the dye (b) direct dyes exhibit
45
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
high levels of color removal that is independent of the number of sulphonate
groups in the dye and (c) reactive dyes exhibit low levels of color removal
Sulfonated reactive group of azo dyes are normally considered to be more
recalcitrant than carboxylated azo dyes The rate limiting step during
decolorization of sulfonated azo dyes is the permeation through the bacterial cell
membrane required for reduction of azo dyes (Lourenco et al 2000 Kodam et
al 2006) It was also observed that the enzyme (azoreductase) production was
related with particular dye structures (Kulla 1981) Some azo dyes are more
resistant to removal by bacterial cells (Bras et al 2001) In the case of the
terminal non-enzymatic reduction mechanism reduction rates are influenced by
changes in an electron density in the region of the azo group causes an increase in
the reduction rate (Walker and Ryan 1971) Hydrogen bonding in addition to the
electron density in the region of the azo bond has a significant effect on the rate of
reduction (Beydilli et al 2000)
236 Electron donor
Literature survey suggests that the oxidation of organic electron donors
andor hydrogen is coupled to the color removal process Recently it was
observed that the addition of electron donors such as glucose or acetate ions
apparently induces the reductive cleavage of azo bonds (Bras et al 2001) The
thermodynamics study shows that the different electron-donating half-reactions
are different due to which the reaction rate is likely to be influenced by the type of
electron donor It was also important to determine the physiological electron
donor for each biological color removal process because it not only induces the
reduction mechanism but also stimulates the enzymatic system responsible for
the reduction process (Van der Zee et al 2001 Pearce et al 2003) It was
observed that the formate acts as a most effective electron donor for the
46
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
anaerobically induced electron transfer pathway to the dye molecule it may be
due to the pathway which shows involvement of formate dehydrogenase enzyme
(Lloyd et al 1997) Yoo et al (2001) found that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye reduction with
the active cells metabolizing the lysis products Certain chemicals such as
thiomersal and p-chloromercuibenzoate inhibit the alcohol dehydrogenase of
NADH-generating systems required to generate reducing equivalents for dye
reduction Therefore the rate of formation of NADH would be rate-limiting
causing inhibition of azo dye reduction (Gingell and Walker 1971)
Coenzyme-reducing equivalents that are involved in normal electron transport by
the oxidation of organic substances may act as the electron donors for azo dye
reduction (Plumb et al 2001)
237 Redox mediator
Rate determining factors for the dye reduction reaction involving the
redox mediator include the redox potential of the mediator in relation to the azo
dye and the specificity of the reducing enzymes Sulfonated group of azo dyes
will pass through the cell membrane where the dye reduction reaction takes
place by using extracellular reducing activity (Keck et al 1997) It was found
that the reducing activity get induced in the presence of redox mediator
compounds such as flavins to shuttle reduction equivalents from the cells to
facilitate the non-enzymatic reduction of the extracellular azo dye (Plumb et al
2001) A very small concentration of the redox mediator is sufficient for this type
of electron transfer Redox mediators are characterized by a redox potential
ranging from minus200 to minus350 mV (Semde et al 1998) The addition of synthetic
electron carriers enhances the rate of reduction of azo dyes by bacterial cells
Quinonendashhydroquinone most widely used as redox mediators (Keck et al 1997)
47
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
The application of natural biodegradable quinones such as lawsone has
technical potential for color removal treatment systems because the reduction rate
is increased without adding any environmentally problematic substances (Rau et
al 2002) Lourenco et al (2001) observed some color removal in the presence
of autoclaved cells suggesting the existence of an active reducing factor that is
capable of dye reduction in the absence of microbial activity
238 Redox potential
It was observed that color removal depends on the redox potential of the
electron donors and acceptors because the rate-controlling step involves redox
equilibrium between the dye and the extracellular reducing agent The redox
potential can be measured with an ease with which a molecule will accept
electrons and can be reduced Therefore the more positive the redox potential
the more readily the molecule get reduced (Bragger et al 1997) The results
suggest that the rate of color removal will increase with increasing (more
positive) half-wave potential of the azo substrate It was reported that there is a
linear relationship between the logarithm of color removal rate and the half-wave
potential of the substrate (Bragger et al 1997) Thus under anaerobic conditions
establishment of low oxidationndashreduction potentials (ltminus400 mV) enhances the
high color removal rates and has an effect on the profile of metabolites that are
generated during the reduction process (Lourenco et al 2001) It was reported
that color removal rate is highest when the redox potential of the system is at its
most negative and the rate falls as the redox potential of the system rises
(Bromley-Challenor et al 2000)
24 Enzymes involved in the decolorization and degradation of dyes
The possible enzymes involved in the diverse biochemical reactions are
present in microorganisms those can efficiently used for the bioremediation to
48
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
treat environmental problems due to their catalytic properties Recently the
enzymatic approach has attracted much interest in the decolorizationdegradation
of the textile and other industrially important dyes from wastewater as an
alternative strategy to conventional physicochemical treatments having inherent
drawbacks The oxidoreductive enzymes are responsible for generating highly
reactive free radicals that undergo a complex series of spontaneous cleavage
reactions It was observed that due to the susceptibility of enzymes to
inactivation by the presence of the other chemicals it is likely that enzymatic
treatment will be the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentration of other
contaminants that may tend to interfere with the enzymatic treatment Mainly the
oxidoreductive enzymes such as lignin peroxidases manganese peroxidases
laccases azoreductases riboflavin reductase and NADH DCIP reductases has
been exploited in the decolorization and degradation of dyes and described in
this section (Das and Reddy 1990 Heinfling et al 1998 Campos et al 2001
Suzuki et al 2001 Nyanhongo et al 2002 Ryan et al 2003 Maier et al
2004)
241 Oxidative enzymes
i) Lignin peroxidase (EC 111114)
Lignin peroxidase (LiP) was first discovered based on the
H2O2-dependent Cα-Cβ cleavage of lignin model compounds and subsequently
shown to catalyze depolymerization of methylated lignin in vitro (Glenn et al
1983 Tien and Kirk 1983 Gold et al 1984) Mainly lignin peroxidases (LiP)
catalyze the oxidation of nonphenolic aromatic lignin moieties and similar
compounds LiP catalyzes several oxidations in the side chains of lignin and
related compounds (Tien and Kirk 1983) by one-electron abstraction to form L
49
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
form reactive radicals (Fig 22) (Kersten et al 1985) Also the cleavage of
aromatic ring structures has been reported (Umezawa and Higuchi 1987) LiP
are glycoproteins with molecular weights estimated at 38-46 kDa LiP has been
used to mineralize a variety of recalcitrant aromatic compounds such as three
Fig 22 Generic scheme of the catalytic cycle of peroxidases (adapted from
Wesenberg et al 2003)
and four-ring PAHs (Guumlnther et al 1998) polychlorinated biphenyls (Krcmaacuter
and Ulrich 1998) and dyes (Chivukula et al 1995) 2-Chloro-1
4-dimethoxybenzene a natural metabolite of white rot fungus is reported to act
as a redox mediator in the LiP-catalyzed oxidations (Teunissen et al 1998)
Recently third group of peroxidases versatile peroxidases (VP) has been
invented in species of Pleurotus and Bjerkandera which that can be considered
as a hybrid between MnP and LiP since they can oxidize not only Mn2+ but also
phenolic and nonphenolic aromatic compounds including dyes (Heinfling et al
1998a b) There are many reviews focused on the molecular biology of white rot
50
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
fungus peroxidases (Martıacutenez 2002 Conesa et al 2002)
ii) Laccase (EC11032)
Laccases are copper containing enzymes belonging to the small group
called as blue oxidase enzymes Laccase also called as the phenol oxidase that
catalyzes the oxidation of several aromatic and inorganic substances
(particularly phenols) with the concomitant reduction of oxygen to water (Duraacuten
et al 2002) (Fig 23) The molecular structure of laccases exhibits four
neighbor copper atoms which are distributed among different binding-sites and
they are classified into three types Copper I II and III which are differentiated
by specific characteristic properties that allow them to play an important role in
the catalytic mechanism of the enzyme (McGuirl and Dooley 1999) The
molecular weight of laccase was observed in the range 60ndash390 kDa
(Reinhammar 1984 Call and Muumlcke 1997) Laccases have been intensively
studied with a focus on their industrial applicability (Yaropolov et al 1994
Bajpai 1999 Gianfreda et al 1999 Rodrıacuteguez et al 1999) molecular genetics
(Cullen 1997 Ong et al 1997 Collins and Dobson 1997) cloning (Hatamoto
et al 1999) and in the degradation of various recalcitrant compounds such as
chlorophenols (Grey et al 1998 Fahr et al 1999) polyaromatic hydrocarbons
(PAHs) (Majcherczyk et al 1998) lignin-related structures (Bourbonnais et al
1996) organophosphorous compounds (Amitai et al 1998) phenols (Bollag et
al 1988 Xu 1996) and last but not least aromatic dyes (Chivukula and
Renganathan 1995 Rodrıacuteguez et al 1999 Kalyani et al 2008 Saratale et al
2009b) Moreover several process using laccases as well as immobilized
laccases have been developed for the treatment of phenolic effluents and
polycyclic aromatic hydrocarbons (Abadulla et al 2000)
51
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
Fig 23 The catalytic cycle of laccases (adapted from Wesenberg et al 2003)
242 Reductive enzymes
i) Azoreductase
Azoreductases are the enzymes which catalyzes the reductive cleavage of
azo bond (-N=N-) to produce colorless aromatic amine products (Chang et al
2001) The azoreductase were observed in many organisms including the rat
liver enzyme cytochrome P-450 rabbit liver aldehyde oxidase and
azoreductases from the intestinal microbiota (Zbaida and Levine 1990 Stoddart
and Levine 1992) Several studies have been reported bacterial cytoplasmic
azoreductases and suggested the application for the purpose of environmental
biotechnology (Zimmermann et al 1982 Rafii and Cerniglia 1993
Moutaouakkil et al 2003 Maier et al 2004 Ramalho et al 2004)
Azoreductases on the basis of their function are categorized as flavin-dependant
azoreductases (Nakanishi et al 2001 Chen et al 2004 Chen et al 2005) and
flavin-independent azoreductases (Blumel et al 2002 Blumel and Stolz 2003)
52
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
The flavin-dependent azoreductases are further organized on the basis of their
cofactor NADH only (Nakanishi et al 2001 Chen et al 2004) NADPH only
(Chen et al 2005) or both (Ghosh et al 1992 Wang et al 2007) as these
coenzymes serve as electron donors Based on the primary amino acid level the
classification of azoreductases is found to be difficult hence recently
classification based on the secondary and tertiary amino acid analysis has been
developed (Abraham 2007)
ii) NADPH-DCIP (Dichorophenol indophenol) reductase
NADH-DCIP reductases are marker enzymes of the bacterial and fungal
mixed function oxidase system and take part in the detoxification of xenobiotic
compounds (Bhosale et al 2006 Parshetti et al 2006 Saratale et al 2007a)
Several studies in our laboratory reported this as a marker enzyme for the
reduction of azo bond because in the presence of this enzyme the 2 6
dichlorophenolindophenol (DCIP) accept an electron from NADH to form its
leuco form Orginally DCIP is a blue in color its oxidized form and becomes
colorless when it is reduced (Fig 24)
O N OCl
Cl
Na+
DCIP (blue)
[H]
[H]
+2 Red
-2 OxidNH OH
Cl
Cl
ONa+
Leuco-DCIP (colorless)
Fig 24 Reduction of DCIP to form its leuco form
iii) Riboflavin reductase
Riboflavin reductase has been reported in the degradation of azo dyes
(Russ et al 2000) In this the non-enzymatic reduction of free flavins by
NADPH and NADH is rather slow requires organism to possess a system to
53
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
catalyze the reaction As a result the enzyme NAD(P)Hflavin oxidoreductase or
flavin reductase are evolved (Ingelman et al 1999) The enzyme flavin
reductase catalyzes the reduction of various flavins that is riboflavin flavin
mononucleotide (FMN) and flavin adenine dinucleotide (FAD) at the expense of
reduced pyridine nucleotides (Spyrou et al 1991) The ability of riboflavin
reductase to act as an azo reductase had been demonstrated the degradation of
scarlet RR and Green HE4BD by the enzyme present in consortium-GR and
Micrococcus glutamicus NCIM-2168 (Saratale et al 2009b and c)
25 Toxicity studies
Variety of synthetic dyestuff released by the textile industries pose a
threat to the environmental safety It is estimated that 10-15 of the dyes is lost
in the effluent during the dyeing process (Robinson et al 2001 Pearce et al
2003) and in the case of reactive azo dyes due to higher solubility up to 50 of
the dye is lost through hydrolysis during the dyeing process which shows
negative aesthetic effect on the wastewater In fact as much as 90 of reactive
dyes could remain unaffected after activated sludge treatment (Patil et al 2008)
The release of textile and dye-house effluent may cause abnormal coloration of
surface waters that creates the problems for both the public and the environment
There are not just artistic problems the greatest environmental concern with the
dyes is their absorption water quality penetration of sunlight and which directly
affects the aquatic flora and fauna In addition the human health impact of dyes
has caused concern for number of years It has been reported that the
contamination of hot chili other spices and baked foods with azo dyes (Calbiani
et al 2004 Mazzetti et al 2004 Wang et al 2007) There is some evidence
that azo dyes have genotoxic effects (Stiborova et al 2002 Stiborova et al
2005 Stiborova et al 2006 An et al 2007) and that ingestion of food products
54
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
contaminated with dyestuffs could lead to exposure in the human gastrointestinal
tract
Toxicity of azo dyes and their metabolic intermediates has been
investigated by many researchers These toxicity studies include genotoxicity
mutagenicity mortality and carcinogenicity diverge from tests with aquatic
fauna (fish algae bacteria etc) to tests with mammals Recently some research
has been carried out to study the effects of dyestuffs and dye containing effluents
on the activity of both aerobic and anaerobic bacteria in wastewater treatment
systems (Pandey et al 2007) Furthermore the occupational exposure to
dyestuffs of human workers in dye manufacturing and dye utilizing industries
has received attention It was observed that purified form of azo dyes are directly
mutagenic and carcinogenic except for some azo dyes (Brown and DeVito
1993) In mammalian system metabolic activation (reduction) of azo dyes is
mainly carried out by bacteria present in the anaerobic parts of the lower
gastrointestinal tract In addition the liver and the kidneys can also reduce the azo
dyes After azo dye reduction in the intestinal tract and kidney the toxic
aromatic amines are absorbed by the intestine and excreted in the urine The
severe effect of aromatic amines is carcinogenesis especially causes bladder
cancer The mechanism of carcinogenicity includes the formation of acyloxy
amines through N-hydroxylation and N-acetylation of the aromatic amines
followed by O-acylation These acyloxy amines can be converted to nitremium
and carbonium ions that bind to DNA and RNA which induces various kinds of
mutations and resulted into tumor formation (Brown and DeVito 1993) In 1975
and in 1982 the International Agency for Research on Cancer (IARC)
summarized the literature on suspected azo dyes mainly amino-substituted azo
dyes fat-soluble azo dyes and benzidine azo dyes but also a few sulfonated azo
55
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
dyes (IARC 1975 1982) Most of the dyes on the IARC list were taken out of
production (Brown and DeVito 1993)
Moreover the aromatic amine with benzidine toluene aniline and
naphthalene moieties causes genotoxicity The toxic effect of these amines
generally depends on the structure or location of the dye molecule Some reports
gives strong evidence about the 2-naphthylamine is a carcinogen whereas
1-naphthylamine is much less toxic (Cartwright 1983) Human exposure to azo
dyes occurs through ingestion inhalation or skin contact Recently some studies
shows that Sudan dyes have genotoxic effects (Stiborova et al 2002 Stiborova
et al 2005 Stiborova et al 2006) From available literature it can be
concluded that almost all textile dyes have genotoxic and adverse environmental
effects
Despite the fact that untreated dyeing effluents might cause serious
environmental and health hazards they are being disposed off in water bodies
and this water is being used for an agriculture purpose (Kalyani et al 2008) Use
of untreated and treated dyeing effluents in the agriculture has direct impact on
the fertility of soil Thus it was of concern to assess the phytotoxicity of the dye
before and after degradation Some investigators studied the toxic effect of azo
dyes and their metabolites in terms of germination rate root and plumule length
as well as the enzymatic system responsible for decolorization present in the
plants (Ghodake et al 2008 Kagalkar et al 2009 Saratale et al 2009a) The
results suggest that after microbial mainly bacterial treatment the extract of
degraded metabolites was found to be less toxic than the parent compound The
foregoing results suggest the potential of utilizing bacterial system to decolorize
textile effluent containing a mixture of textile dyes via appropriate bioreactor
operations
56
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
26 Decolorization and degradation of azo dyes using immobilized cell
system
Several studies reported that immobilized-cell systems which not only
increase the biomass concentration but also enhance the stability mechanical
strength and reusability of the biocatalyst Cell immobilization by entrapment
within natural or synthetic matrices is particularly suitable for bacterial
decolorization of azo dyes since it creates a local anaerobic environment
favorable to oxygen-sensitive decolorization In contrast despite the fact that the
suspended-cell system allows better contact with the substrates it may be less
feasible in practical applications due to the requirement of downstream
solid-liquid separation and the difficulty of achieving a high cell density Due to
these reasons few researchers have reported utilization of immobilized-cell
systems for decolorization of wastewater and most cases have focused on
immobilization of fungal biomass (Chang et al 2001 Chen et al 2005) rather
than bacterial cells which also hold potential for decolorization (Yang et al
1996 Palleria et al 1997) In fact it has been demonstrated that immobilized
microbial systems greatly improve bioreactor efficiency for instance increasing
process stability and tolerance to shock loadings allowing higher treatment
capacity per unit biomass and generating relatively less biological sludge
(Marwaha et al 1998 Zhang et al 1999) In addition biocatalyst
immobilization could significantly increase the entrapped biomass concentration
and thereby reduces the bioreactor volume to satisfy a critical criterion in
practical uses Cell immobilization with gel entrapment holds an extra benefit of
creating a local anaerobic environment which is particularly suitable for
oxygen-sensitive bacterial decolorization (Palleria et al 1997) Therefore it
seems promising to use immobilized cell system to develop decolorization
57
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
bioprocesses
Considering the overview of the literature the present study was
undertaken to determine the potential of developed consortium-GR (consisting
of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168) and
individual strains for the degradation of various azo dyes The present work was
accomplished considering the following objectives
27 Objectives of the thesis
1 To design a microbial consortium of efficient microorganisms for the
degradation of Scarlet RR Green HE4BD and Navy Blue HE2R
2 To standardize the physicochemical conditions as well as different carbon
and nitrogen source for the efficient decolorization
3 To use this developed consortium for the decolorization and degradation of
mixture of different industrial dyes
4 To study the nature of enzyme system responsible for the decolorization and
degradation of azo dyes and possible use for co metabolism
5 To study of longetivity of decolorization activity of microorganisms
enzymes and immobilized cells in repeated batch decolorization tests
6 To study the mineralization change in the individual dye and mixture of dyes
during decolorization process by using COD and TOC also to perform the
toxicity studies using phytotoxicity and microbial toxicity study
7 To determine the structural configuration of metabolites formed using
decolorization process by using analytical techniques viz Uv-Vis ADMI
(for mixture of dyes) FTIR HPLC and GCMS
8 To study the metabolic pathway involved in dye degradation by using
enzymatic and analytical results
58
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59
Review of literature
9 To develop the immobilization methods by using different gel matrices and
to study their potential using different azo dyes by optimizing
physicochemical conditions
10 To design a fixed bed reactor (FBR) using calcium alginate immobilized
beads and using agricultural raw material as a biofilm to treat individual and
mixture of various industrial dyes
59