document1

Biodegradation of Synthetic Dyes—A Review

Hazrat Ali

Received: 27 September 2009 /Accepted: 26 February 2010 /Published online: 30 March 2010# Springer Science+Business Media B.V. 2010

Abstract The contamination of soils and waters bydye-containing effluents is of environmental concern.Due to the increasing awareness and concern of theglobal community over the discharge of syntheticdyes into the environment and their persistence there,much attention has been focused on the remediationof these pollutants. Among the current pollutioncontrol technologies, biodegradation of synthetic dyesby different microbes is emerging as an effective andpromising approach. The bioremediation potentials ofmany microbes for synthetic dyes have been demon-strated and those of others to be explored in future.The biodegradation of synthetic dyes is an economic,effective, biofriendly, and environmentally benignprocess. Bioremediation of xenobiotics includingsynthetic dyes by different microbes will hopefullyprove a green solution to the problem of environmen-tal soil and water pollution in future. This reviewpaper discusses comprehensively the science and artsof biodegradation of synthetic dyes.

Keywords Biodegradation . Bioremediation .

Decolorization .Microbial . Synthetic dyes

1 Introduction

Environmental pollution is one of the major and mostimportant problems of the modern world. Develop-ment in agriculture, medicine, energy sources, and allchemical industries is necessary in order to fulfill theneeds and demands of the overgrowing humanpopulation. Almost all processes employed by manfor the production of goods and services lead to theproduction of environmental pollutants. These pollu-tants are released into air, water, and soil and havedetrimental effects on the health of humans, plants,animals, and microbes. Human endeavors for theproduction and improvement of goods and servicescannot be absolutely stopped because these areneeded by humans for their survival on earth.Alternatively, we must look for green processes—processes that lead to the production of environmen-tally friendly products. We must also focus ourattention on ways for the eradication and reductionof the existing environmental pollution. Thus, for asustainable human society, we need green chemistryand environmental remediation. Over the last twodecades, there has been a tremendous increase inawareness of the toxic and carcinogenic effects ofmany polluting chemicals that were not consideredhazardous in the past (King et al. 1997). Unlike thenaturally occurring organic compounds that arereadily degraded upon introduction into the environ-ment, some of the synthetic chemicals are extremely

Water Air Soil Pollut (2010) 213:251–273DOI 10.1007/s11270-010-0382-4

H. Ali (*)Department of Biotechnology, University of Malakand,Dir Lower,Chakdara, NWFP, Pakistane-mail: [email protected]

resistant to biodegradation by native microorganisms(Fernando and Aust 1994). Many of the recalcitrantcompounds are major environmental pollutants, suchas munitions waste, pesticides, organochlorines, poly-chlorinated biphenyls, polycyclic aromatic hydrocar-bons, wood preservatives, synthetic polymers, andsynthetic dyes (Pointing 2001). Most synthetic dyesare toxic and highly resistant to degradation due totheir complex chemical structures (Lu et al. 2009).

Synthetic dyes are extensively used in textiledyeing, paper printing, color photography, pharma-ceutical, food, cosmetic, and leather industries (Rafiiet al. 1990; Kuhad et al. 2004; Couto 2009). Since1856, over 105 different dyes have been producedworldwide with an annual production of over 7×105

metric tons (Chen et al. 2003). Paper and pulp mills,textiles and dyestuff industries, distilleries, andtanneries are some of the industries, which releasehighly colored wastewaters (Raghukumar 2000). Thetextile industry is one of the greatest generators ofliquid effluent pollutants due to the high quantities ofwater used in the dyeing processes (Kalyani et al.2009). The traditional textile finishing industry con-sumes about 100 L of water to process about 1 kg oftextile materials (Couto 2009). All dyes do not bind tothe fabric; depending on the class of the dye, its lossin wastewaters could vary from 2% for basic dyes toas high as 50% for reactive dyes, leading to severecontamination of surface and ground waters in thevicinity of dyeing industries (O’Neill et al. 1999). Itis estimated that 280,000 tons of textile dyes aredischarged in textile industrial effluent every yearworldwide (Jin et al. 2007). Effluents from the textileindustries containing dyes are highly colored andare therefore visually identifiable (Kilic et al. 2007).Color is usually the first contaminant to be recognizedin wastewater (Wong and Yu 1999), and thus, thecolor present in dye effluents gives a straight forwardindication of water being polluted, and the dischargeof these highly colored effluents can damage directlythe receiving waters (Chen et al. 2003). Due to large-scale production and extensive application, syntheticdyes can cause considerable environmental pollutionand are serious health-risk factors (Forgacs et al.2004). The untreated dyeing effluents that arestraightly used in agriculture have a serious impacton environment and human health (Pourbabaee et al.2006). Disposal of the untreated dyeing effluent,without any treatment, in water bodies causes serious

environmental and health hazards (Shedbalkar et al.2008). Industrial effluents containing synthetic dyesreduce light penetration in receiving water bodies andthus affect the photosynthetic activities of aquaticflora, thereby badly affecting the food source ofaquatic organisms. The thin layer of discharged dyesformed over the surface of a receiving water bodyalso decreases the amount of dissolved oxygen in thewater, thereby affecting the aquatic fauna. Further-more, dye-containing effluents increase biochemicaloxygen demand of the contaminated water (Annuar etal. 2009). Many dyes are visible in water at concen-trations as low as 1 mg L−1 (Pandey et al. 2007).Thus, apart from affecting the health of plants andanimals, synthetic dyes are also undesirable in waterbodies from aesthetic point of view. Of all knowndyestuffs in the world, azo dyes make up about a half,making them the largest group of synthetic colorantsand the most common synthetic dyes released into theenvironment (Zhao and Hardin 2007). The fast coloreddyes are a major source of concern to environmen-talists, since such pollutants, besides causing aestheticdamage to sites, are also toxic and carcinogenic(Meyer 1981). In recent years, interest in environmen-tal control of dyes has increased, due to their possibletoxicity and carcinogenicity; this is because many dyesare comprised of known carcinogens, such as benzi-dine and other aromatic compounds (Singh 2006).There is a great need to develop an economic andeffective way of dealing with the textile dyeing wastein the face of the ever-increasing production activities(Park et al. 2007). Government legislation is becomingmore and more stringent, especially in more developedcountries, regarding the removal of dyes from indus-trial effluents (Robinson et al. 2001; Kuhad et al.2004). Over the last decade, azo dyes that couldbreakdown to carcinogenic aromatic amines have beenlargely phased out in Europe (Pinheiro et al. 2004).

Different methods are available for the remediationof dye wastewaters. These include physicochemicalmethods, like adsorption, chemical oxidation, precipi-tation, coagulation, filtration, electrolysis, photodegra-dation, and biological, and microbiological methods.Adsorption on activated carbon is an effective methodfor the removal of color, but it is too expensive (Fu andViraraghavan 2001). The major disadvantage of phys-icochemical methods has been largely due to the highcost, low efficiency, limited versatility, interference byother wastewater constituents, and the handling of the

252 Water Air Soil Pollut (2010) 213:251–273

waste generated (van der Zee and Villaverde 2005;Kaushik and Malik 2009). Traditional wastewatertreatment technologies have proven to be markedlyineffective for handling wastewater of synthetic textiledyes because of the chemical stability of thesepollutants (Forgacs et al. 2004). The development ofefficient and environmentally friendly technologies todecrease dye content in wastewater to acceptable levelsat affordable cost is of utmost importance (Couto2009). Biological methods are generally consideredenvironmentally friendly as they can lead to completemineralization of organic pollutants at low cost(Pandey et al. 2007). It is now known that severalmicroorganisms, including fungi, bacteria, yeasts, andalgae, can decolorize and even completely mineralizemany azo dyes under certain environmental conditions(Pandey et al. 2007). The use of microorganisms forthe removal of synthetic dyes from industrial effluentsoffers considerable advantages; the process is relativelyinexpensive, the running costs are low, and the endproducts of complete mineralization are not toxic(Forgacs et al. 2004). Thus, biodegradation is apromising approach for the remediation of syntheticdyes wastewater because of its cost effectiveness,efficiency, and environmentally friendly nature (Vermaand Madamwar 2003; Jirasripongpun et al. 2007;Shedbalkar et al. 2008; Gopinath et al. 2009). As abest alternative, much interest is now focused onbiodegradation of dyes (McMullan et al. 2001; An etal. 2002). Bioremediation may be the most effectivemethod of treating industrial dyes wastewater (Nozakiet al. 2008).

The growing publication of research and reviewarticles dealing with the remediation of environmentalpollution caused by synthetic dyes is an indicationand proof of the global concern over this issue.During the last two decades, the scientific communityin the whole world and especially in India and Chinahas been active in research on the problems caused bythis source of environmental pollution and its effec-tive remediation. This review paper is an effort todiscuss the science and arts of biodegradation ofsynthetic dyes.

2 Structures of Dyes

The major structure element responsible for lightabsorption in dye molecules is the chromophore group,

i.e., a delocalized electron system with conjugateddouble bonds. Chromophores frequently contain heter-oatoms as N, O, and S, with non-bonding electrons.Common classes of dyes, based on the chromophorepresent, are shown in Table 1 (Ramalho 2005).

3 Microbial Decolorization of Dyes

Decolorization of dyes may take place in twoways: either adsorption on the microbial biomass orbiodegradation of the dyes by the cells (Zhou andZimmermann 1993). Adsorption of dyes may occuron growing/living microbial cells as well as on deadmicrobial cells. The adsorption in this case is referredto as biosorption because it occurs on a biomaterial.In case of biosorption, the original structure of thedyes remains intact, i.e., not degraded into fragments.In contrast, in biodegradation, the original dyestructure is destroyed, and the pollutant is split intofragments by the microbial cells, sometimes achievingcomplete mineralization, i.e., conversion of the xenobi-otic into CO2, H2O, and some salts of inorganic origin.From the very nature of the two processes (biosorptionand biodegradation), biodegradation seems more natu-ral in its operation. Biosorption of dyes does noteradicate the problem because the pollutant is notdestroyed but instead entrapped into the matrix of theadsorbent (the microbial biomass). The disposal of themicrobial biomass containing adsorbed dyes itself is abig hurdle in their proposed role in biocleaning ofcolored waters (Chander and Arora 2007). Thus,biosorption may not be a practical approach fortreating large volumes of dye-contaminated industrialeffluents because of the problems associated withdisposal of the large volumes of biomass afterbiosorption of dyes from industrial effluents (Kuhadet al. 2004). However, in addition to biodegradation, abiosorption mechanism might also play an importantrole in the decolorization of dyes by living fungi(Fu and Viraraghavan 2001), and the biosorption ofdyes may be of interest in biorecovery of thesesynthetic chemicals from spent dye baths. This ispossible through desorption of the adsorbed dyes usingsuitable solvents or solvent mixtures. This reviewpaper is a focus on biodegradation of synthetic dyes,as this process has the potential to eradicate theproblem of the existence of organic xenobiotics in theenvironment and the related pollution.

Water Air Soil Pollut (2010) 213:251–273 253

Table 1 Classification of dyes based on the chromophore present

Class Chromophore Example

OH

NO2

NO2C.I. Acid Yellow 24

N

O

O

Nitro dyes

NO

NO

ON OH

Fast Green O

N ONitroso dyes

CH3N

CH3 NNNaO3SNNAzo dyes

Methyl Orange

NMe2

NMe2Me2N

CTriphenyl-methane dyes

C.I. Basic Violet 3

O

C

C

O

HO OH

C

O

C

O

OPhthalein dyes

PhenolphthaleinH

N

SO3NaO

N

O

H

NaO3S

N

N

H

OH

OIndigo dyes

C.I. Acid Blue 71NH2

NH

SO2CH2CH2OSO2OH

O

O

Anthraquinone dyes

O

O

C.I. Reactive Blue 19

SO2OH


4 Biodegradation of Dyes

Biodegradation is defined as the biologically medi-ated breakdown of chemical compounds; it is anenergy-dependent process and involves the break-down of dye into various byproducts through theaction of various enzymes (Kaushik and Malik2009). Biodegradation of synthetic dyes not onlyresults in decolorization of the dyes but also infragmentation of the dye molecules into smaller andsimpler parts (breakdown products). Decolorizationof the dye occurs when the chromophoric center ofthe dye is cleaved (Kaushik and Malik 2009).Various microorganisms, including fungi, bacteria,yeasts, and algae, have been used for decolorizationand degradation of synthetic dyes. They have beenshown to have different capabilities for degradingdifferent dyes. Among the different groups of micro-organisms used for biodegradation of synthetic dyes,some have specific advantages over others. Theeffectiveness of microbial decolorization dependson the adaptability and the activity of the selectedmicroorganisms (Chen et al. 2003). Development ofefficient dye degradation biotechnology requiresapplication of a suitable selected strain and its useunder favorable conditions to realize the degradationpotential (Novotny et al. 2004b).

4.1 Biodegradation of Dyes by Fungi

The single class of microorganisms most efficient inbreaking down synthetic dyes is the white-rot fungi(Couto 2009). White-rot fungi are a class of micro-organisms that produce efficient enzymes capable ofdecomposing dyes under aerobic conditions. Theyproduce various oxidoreductases that degrade ligninand related aromatic compounds (Nozaki et al. 2008).In addition to their natural substrate, white-rot fungihave been found to be capable of mineralizing adiverse range of persistent organic pollutants, whichdistinguishes them from biodegradative bacteria thattend to be rather substrate specific (Reddy 1995). Theability of ligninolytic fungi to oxidize a wide varietyof organic pollutants including synthetic dyes is dueto an extracellular non-specific and non-stereoselectiveenzyme system consisting of lignin peroxidases (LiP),manganese peroxidases (MnP) and laccases (Kuhadet al. 2004; Enayatzamir et al. 2009; Couto 2009). The

fungal mycelia have an additive advantage over single-cell organisms by solubilizing the insoluble substratesby producing extracellular enzymes (Kaushik andMalik 2009). White-rot fungi are the most intensivelystudied dye-decolorizing microorganisms; because oftheir nonspecific lignin-modifying enzymes, thesefungi are able to transform a wide range of organiccompounds (Wesenberg et al. 2003; Novotny et al.2004a). This group of microorganisms is central to theglobal carbon cycle as a result of their ability tomineralize the complex polymeric woody plant mate-rial lignin (Kuhad et al. 2004). Fungi, due to theirexcretion of extracellular enzymes, are known to beable to degrade—though possibly not completely—structures that are difficult for bacteria to handle (Forssand Welander 2009). Wood-rotting microorganismsmay be good candidates for biodegradation of syntheticdyes. This is because lignocellulose-containing materialsconsist partly of complex molecules with similarstructures as textile dyes, which might make the micro-organisms growing on these materials adapted torefractory organic compounds (Forss and Welander2009). Table 2 presents a summary of some studieson biodegradation of dyes by fungi.

4.2 Biodegradation of Dyes by Bacteria

In comparison to fungal decolorization, bacterialdecolorization is normally faster (Kalyani et al.2009). It is well known that bacteria degrade azodyes reductively under anaerobic conditions to color-less aromatic amines. The carcinogenicity of an azodye may be due to the dye itself or aryl aminederivatives produced during the reductive biotransfor-mation of an azo linkage (Dawkar et al. 2009). Thesecolorless aromatic amines should be degraded furtherbecause these may be toxic, mutagenic, and carcino-genic to humans and animals (Chen 2006). Aromaticamines formed during anaerobic cleavage of theazo dyes could be further degraded in an aerobictreatment system (Kuhad et al. 2004; van der Zee andVillaverde 2005). According to the concept ofcombined anaerobic–aerobic treatment, azo dyesshould be removed from the water phase by (anaerobic)reduction followed by (aerobic) oxidation of the dyes’constituent aromatic amines, and this treatment holdspromise as a method to completely remove azo dyesfrom wastewater (van der Zee and Villaverde 2005).


Table 2 Biodegradation of synthetic dyes by fungi

Fungi Dye (conc.) Decolorization (%) Incubation Period References

Acremonium kiliense Malachite green (5 mg L−1) 95.4 72 h Youssef et al. (2008)

Aspergillus flavus Malachite green (50 µM) 97.43 6 days Ali et al. (2009)

Alternaria solani Malachite green (50 µM) 96.91 6 days Ali et al. (2009)

Aspergillus niger Reactive red 120 (40 mg L−1) 74.2 4 days Husseiny (2008)Direct red 81 (10 mg L−1) 78.3 4 days

Coriolus versicolor Acid Orange II (100 mg L−1)a 55 1 day Hai et al. (2008)Poly S119 15 1 day

Funalia trogii Reactive blue 19 (100 mg L−1)a 96.3 8 h Park et al. (2007)Reactive blue 49 100 8 h

Acid violet 43 96.2 8 h

Reactive black 5 78.3 24 h

Reactive orange 16 22.2 24 h

Acid black 52 59 24 h

Fusarium solani Crystal violet (2.5 mg L−1)a 98 2 days Abedin (2009)Malachite green 96 2 days

Irpex lacteus Reactive orange 16 (150 mg L−1)a 85.8 7 days Novotny et al. (2004a)Remazol brilliant blue R 99.7 7 days

Irpex lacteus 238 Reactive orange 16 (200 mg L−1)a 95.8 14 daysb Novotny et al. (2004b)Congo red 55.8

Reactive black 5 98.4

Naphthol blue black 99.1

Chicago Sky Blue 98.6

Remazol brilliant blue R 95.1

Disperse blue 3 94

Methylene blue 80.3

Cu-phthalocyanine 96.7

Bromophenol blue 96

Lentinus polychrous Indigo carmine (20 mg L−1)a 97.6 3 h Sarnthima et al. (2009)Remazol brilliant blue R (RBBR) 57.6 1 h

Bromophenol blue 36.7 16 h

Methyl red 38.0 16 h

Penicillium ochrochloron Cotton blue (50 mg L−1) 93 2.5 h Shedbalkar et al. (2008)

Pycnoporus sanguineus Trypan blue (20 mg L−1) 70 24 h Annuar et al. (2009)

Thelephora sp. Orange G (50 µM)a 33.3 9 days Selvam et al. (2003)Congo red 97.1 8 h

Amido black 10B (25 µM) 98.8 24 h

Trametes sp. SQ01 Bromophenol blue (100 mg L−1)a 100 7 days Yang et al. (2009)Fast blue RR ∼100 4 days

Congo red ∼100 5 days

Amido black 10B ∼100 6 days

Orange G ∼100 6 days

Remazol brilliant blue R (200 mg L−1) ∼100 7 days

Crystal violet (15 mg L−1) 37 7 days

Trametes versicolor Amaranth (33 mg L−1) 100 3.5 h Ramsay and Nguyen (2002)Congo red (30.5 mg L−1) 100 22 h

a Concentration of all dyes used in a given study is the same unless otherwise indicatedb Incubation period in a given study is the same unless otherwise indicated


Table 3 presents a summary of some studies onbiodegradation of dyes by bacteria.

4.3 Biodegradation of Dyes by Yeasts

Very little work has been devoted to study thedecolorization ability of yeast (Kuhad et al. 2004;

Jadhav et al. 2008; Saratale et al. 2009b). Comparedto bacteria and filamentous fungi, yeasts have manyadvantages; they not only grow rapidly like bacteria,but like filamentous fungi, they also have the abilityto resist unfavorable environments (Yu and Wen2005). Table 4 presents a summary of some studieson biodegradation of dyes by yeasts.

Table 3 Biodegradation of synthetic dyes by bacteria

Bacteria Dye (conc.) Decolorization (%) Incubation period References

Aeromonas hydrophila Crystal violet (50 mg L−1)a >90 10 h Ren et al. (2006)Basic fuchsine >90 10 h

Brilliant green >90 10 h

Malachite green >90 10 h

Acid amaranth >85 36 h

Great red GR >85 36 h

Reactive red KE-3B >85 36 h

Reactive brilliant blue K-GR >85 36 h

Aeromonas hydrophila Reactive red 198 (3000 mg L−1) >90 8 days Chen et al. (2003)

Bacillus sp. VUS Navy blue 2GL (50 mg L−1) 94 48 h Dawkar et al. (2009)

Citrobacter sp. Crystal violet (5 µM)a 100 1 hb An et al. (2002)Gentian violet 100

Malachite green 100

Brilliant green 100

Basic fuchsine 98

Methyl red 100

Congo red 65

Enterobacter cloacae Reactive black 5 (1000 mg L−1) 35.63 120 h Wang et al. (2009)

Enterobacter sp. Reactive red 195 (30 mg L−1) 91 2 days Jirasripongpun et al. (2007)

Yersinia sp. Reactive red 195 (30 mg L−1) 87 2 days Jirasripongpun et al. (2007)

Serratia sp. Reactive red 195 (30 mg L−1) 46 2 days Jirasripongpun et al. (2007)

Enterococcus gallinarum Direct black 38 (20–250 mg L−1) 71–85 24 h Bafana et al. (2008)

Kocuria rosea Malachite green (50 mg L−1) 100 5 h Parshetti et al. (2006)

Micrococcus glutamicus Scarlet R (150 mg L−1) 100 36 h Saratale et al. (2009a)

Nocardia corallina Crystal violet (2.3 µM) 100 24 h Yatome et al. (1993)

Proteus vulgaris Scarlet R (150 mg L−1) 100 30 h Saratale et al. (2009a)

Pseudomonas putida Crystal violet (60 µM) ∼80 7 days Chen et al. (2007)

Pseudomonas sp. SUK1 Reactive red 2 (1000 mg L−1) 95 18 h Kalyani et al. (2009)

Shewanella decolorationis Crystal violet (50 mg L−1) ∼100 28 h Chen et al. (2008)

Shewanella putrefaciens Reactive black 5 (100 mg L−1)a 100 6 h Khalid et al. (2008)Direct red 81 100 8 h

Acid red 88 100 8 h

Disperse orange 3 100 8 h



4.4 Biodegradation of Dyes by Algae

Some groups of microorganisms have not been yetfully studied for their abilities to degrade syntheticdyes and other organic xenobiotics. For example,cyanobacteria (blue-green algae) have an ubiquitousdistribution, but information about their role infunctioning of ecosystems, including degradation ofrecalcitrant compounds such as dye and dyestuffs, isscanty (Semple et al. 1999). Table 5 presents a summaryof some studies on biodegradation of dyes by algae.

5 Biodegradation of Dyes by Mixed MicrobialCultures

With increasing complexity of a xenobiotic, it cannotbe expected to find complete catabolic pathways in asingle organism; a higher degree of biodegradationand even mineralization can be expected and accom-plished when cometabolic activities within a micro-bial community complement each other (Knackmuss1996; Nigam et al. 1996). The treatment systemscomposed of mixed microbial populations possess

Table 4 Biodegradation of synthetic dyes by yeasts

Yeasts Dye (conc.) Decolorization (%) Incubationperiod (h)

References

Candida krusei Reactive brilliant red K-2BP (50 mg L−1)a 98 24b Yu and Wen (2005)Weak acid brilliant red B 94

Reactive black KN-B 96

Acid mordant yellow 72

Reactive brilliant blue X-BR 78

Acid mordant red S-80 72

Acid mordant light blue B 93

Reactive turquoise blue KN-G 62

Galactomyces geotrichum Methyl red (100 mg L−1) 100 1 Jadhav et al. (2008)Malachite green (50 mg L−1)a 97 9

Scarlet RR 100 18

Orange HE 4B 75 18

Amido black 10B 92 18

Pseudozyma rugulosa Reactive brilliant red K-2BP (50 mg L−1)a 99 24b Yu and Wen (2005)Weak acid brilliant red B 98

Reactive black KN-B 96

Acid mordant yellow 94

Reactive brilliant blue X-BR 85

Acid mordant red S-80 22

Acid mordant light blue B 89

Reactive turquoise blue KN-G 67

Trichosporon beigelii Navy blue HER (50 mg L−1)a 100 24b Saratale et al. (2009b)Red HE7B 85

Golden yellow 4BD 60

Green HE 4BD 70

Orange HE2R 50

Malachite green 90

Crystal violet 57

Methyl violet 73



higher degree of biodegradation and mineralizationdue to synergistic metabolic activities of microbialcommunity and offer considerable advantages overthe use of pure cultures in the degradation of syntheticdyes (Khehra et al. 2005). In most studies, themicrobial consortia have been found more effectivethan pure cultures (Kuhad et al. 2004). The individualstrains (of a microbial consortium) may attack the dyemolecule at different positions or may use decompo-sition products produced by another strain for furtherdecomposition (Forgacs et al. 2004). Thus, thebiodegradation of dyes may be enhanced using mixedmicrobial cultures due to synergistic effect. A micro-bial consortium consisting of both fungi and bacteriamay be efficient in biodegradation and mineralizationof synthetic dyes and other organic xenobiotics. Thisis because fungi can initiate the degradation ofrecalcitrant compounds (which are not amenable tobacterial degradation); however, to complete the totalorganic carbon (TOC) removal, bacteria are alsorequired (Hai et al. 2008). Mixed culture consortiathat are capable of surviving in effluents by utilizingthe constituents as sources of carbon, energy, andnitrogen would make the process economicallyfeasible (Kuhad et al. 2004).

Dafale et al. (2008) used a microbial consortiumconsisting of Pseudomonas aeruginosa, Bacilluscirculans, and some other unidentified laboratoryisolates (NAD1 and NAD6) for the decolorization ofReactive Black 5 and other azo dyes. The consortiumdecolorized 100 mg L−1 Reactive Black 5 about 90%and 1,000 mg L−1 Reactive Black 5 about 70% after anincubation period of 48 h. The percent decolorization ofother azo dyes Reactive Red 11, Reactive Red 141,Reactive Violet 13, Reactive Orange 16, Direct Green6, Acid Orange 7, and Acid Yellow 36 at a concentra-

tion of 100 mg L−1 was 84, 86, 90, 88, 60, 65, and 41,respectively, after an incubation period of 48 h.

6 Biodegradation of Dyes by Immobilized Cells

Recently immobilized microbial cells have been usedfor biodegradation of synthetic dyes. Cell immobiliza-tion is of two types, i.e., entrapment and attachment. Inentrapment, the microorganisms are entrapped in theinterstices of fibrous or porous materials, while inattachment, the microorganisms adhere to surfaces orother organisms (Couto 2009). Immobilized culturestend to have a higher level of activity and are moreresilient to environmental perturbations, such as pH orexposure to toxic chemicals concentrations, thansuspension cultures (Kuhad et al. 2004; Couto 2009).Zhang et al. (1999) studied the decolorization of theazo dye Orange II by free and alginate-immobilizedcells of an unidentified white-rot fungus. They foundthat the immobilized fungus performed better than thefree one and could continuously be reused for morethan 2 months with high efficiency (97% in 24 h). Cellimmobilization by entrapment within natural or syn-thetic matrices is particularly suitable for bacterial dyedecolorization, since it creates a local anaerobicenvironment favorable to dye metabolism (Stolz 2001).

7 Biodegradation of Dyes by Microbial Fuel Cells

The use of microbial fuel cells (MFCs) for biodegra-dation of synthetic dyes is an emerging research area.To date, very little work has been reported onbiodegradation of dyes using microbial fuel cells.Recently, microbial fuel cells have demonstrated the

Table 5 Biodegradation of synthetic dyes by algae

Algae Dye (conc.) Decolorization (%) Incubationperiod (days)

References

Chroococcus minutus Amido Black 10B (100 mg L−1) 55 26 Parikh and Madamwar (2005)

Gloeocapsa FF Sky Blue (100 mg L−1)a 90 26b Parikh and Madamwar (2005)pleurocapsoides Acid Red 97 83

Phormidium ceylanicum FF Sky Blue (100 mg L−1)a 80 26b Parikh and Madamwar (2005)Acid red 97 89



ability to simultaneously produce energy and enhancethe degradation of some biorefractory contaminants(Luo et al. 2009; Morris et al. 2009). MFCs may offera new technique in practical applications for enhanceddecolorization of azo dyes while at the same timerecovering electricity from a readily degradableorganic carbon source (Sun et al. 2009). Sun et al.(2009) investigated decolorization of Active BrilliantRed X3 (ABRX3) in the air-cathode single-chamberMFC at an external resistance of 500 Ω at an initialABRX3 concentration of 300 mg L−1 using glucoseas a co-substrate. According to them, accelerateddecolorization of ABRX3 was observed in the air-cathode single-chamber MFC as compared to a tradi-tional anaerobic reactor at any given time during the 48 hdecolorization experiment. The color was removedalmost completely after 48 h in the MFC, while 80.1%was removed in the traditional anaerobic reactor andonly 11.2% in the abiotic control (autoclaved sludge).Electricity generation from glucose was not significantlyaffected by ABRX3 at 300 mg L−1, while higherconcentrations inhibited the electricity generation dueto the competition between the azo dye and the anodefor electrons from carbon sources. The use of microbialfuel cells for biodegradation of synthetic dyes and otherorganic pollutants may open new horizons in bioelec-tricity and energy research.

8 Factors Affecting Biodegradation of Dyes

Ecosystems are dynamic environments with variableabiotic conditions, like pH, temperature, presence ofoxygen, metals, salts, etc. Microorganisms, which havea key role in the global C, N, and S cycles, are affectedby changes in these parameters, and consequently, theirdecomposing activities are also affected. Thus, whileevaluating the potential of different microorganismsfor degrading particular organic xenobiotics, the effectsof these parameters are to be taken into account.Optimization of such abiotic conditions will greatly helpin the development of industrial-scale bioreactors forbioremediation. In this section, some of the factorsaffecting biodegradation of synthetic dyes are discussed.

8.1 pH

Generally, fungi and yeasts show better decolorizationand biodegradation activities at acidic or neural pH

while bacteria at neutral or basic pH. Nozaki et al.(2008) studied the decolorization of 27 different dyes,including monoazo, diazo, phthalocyanine, and tri-phenylmethane dyes, using 21 different basidiomy-cetes. They found that the optimum pH for thedecolorization of the dyes was 3.0–5.0. Jadhav et al.(2008), while studying the biodegradation of methylred by Galactomyces geotrichum (yeast) found thatthis strain could completely decolorize methyl red atpH range of 3–5. The optimum pH for decolorizationwas 3. Sarnthima et al. (2009) studied decolorizationof synthetic dyes by white-rot fungus Lentinuspolychrous. They found that the optimum pH fordecolorization of Indigo Carmine, Remazol BrilliantBlue R (RBBR), Bromophenol Blue and Methyl Redwas 9.0, 3.0, 4.0, and 4.0–5.0, respectively. Accordingto Yang et al. (2009), the optimum pH for decoloriza-tion of Remazol brilliant blue R (200 mg L−1) by awhite-rot fungus Trametes sp. SQ01 was 4.5. Similarly,according to Shedbalkar et al. (2008), the optimumpH for decolorization of Cotton Blue (50 mg L−1)by Penicillium ochrochloron MTCC 517 was 6.5.Raghukumar et al. (1996) reported the effect of pH oncolor removal by three marine fungi, and the effectivepH was 4.5. Saratale et al. (2009b), while studying theeffect of pH on decolorization and biodegradation ofNavy Blue HER by Trichosporon beigelii (yeast)found that the rate of decolorization was optimum atpH7. These studies show that fungi and yeasts degradesynthetic dyes mostly at low or acidic pH. Kaushik andMalik (2009) also state that for majority of the fungi,the optimum pH for dye decolorization lies in theacidic range. They point out that the fungal ligninolyticenzymes show maximal activity at low pH; therefore,efficient dye decolorization is also observed at low pH.Furthermore, fungi can grow at low pH, normallyranging from 4 to 5 (Fu and Viraraghavan 2001).

Wang et al. (2009) studied decolorization ofReactive Black 5 by a bacterial strain Enterobactersp. EC3. According to their results, Enterobacter sp.EC3 showed a high decolorization rate at pH7.0 after108 h of incubation. Similar decolorization efficiencywas observed from pH8.0–12.0 in 120 h, whereas therate of color removal was much lower at acidicconditions (pH4.0 and 6.0). The authors concludedthat this could be due to the fact that the optimum pHfor the growth of Enterobacter sp. EC3 was neutral.Saratale et al. (2009a) studied decolorization andbiodegradation of Scarlet R by a microbial consortium-


GR consisting of two bacterial strains, Proteus vulgarisNCIM-2027 andMicrococcus glutamicus NCIM-2168.They found that percent decolorization of Scarlet R atpH5, 6, 7, and 8 was 62, 82, 100, and 100 after 24, 24,14, and 36 h, respectively, by P. vulgaris while 55, 65,100, and 100 after 24, 24, 20, and 48 h, respectively,byM. glutamicus. From these results, it is clear that thefavorable pH for dye decolorization by these bacterialstrains was 7–8 with optimum pH being 7.

8.2 Temperature

Temperature is an important environmental factor, andthe biodegradation activities of microorganisms areaffected by changes in temperature. It is a commonobservation that dead animal bodies are decomposedfaster in summer than in winter. It is because, insummer, the warmer environment is favorable for thegrowth and multiplication of the decomposers (mostlysoil bacteria and fungi). However, this relationship isnot linear beyond a certain temperature (optimumtemperature for the growth and reproduction of theconcerned microorganisms). Beyond the optimumtemperature, the degradation activities of the micro-organisms decrease because of slower growth andreproduction rate and deactivation of enzymes respon-sible for degradation. Thus, the biodegradation perfor-mance of microorganisms will be best at the optimumtemperature needed for their growth, reproduction, andactivities.

Different fungi have different optimum growthtemperatures, with most of them growing at 25–35°C(Fu and Viraraghavan 2001). According to Shedbalkaret al. (2008), the optimum temperature for decoloriza-tion of Cotton Blue by P. ochrochloron MTCC 517was 25°C. Jadhav et al. (2008) studied decolorizationof Methyl Red by G. geotrichum (yeast) at differenttemperatures, i.e., 5°C, 30°C, and 50°C. The optimumtemperature for decolorization was found to be 30°C.According to Saratale et al. (2009b), when thedecolorization of Navy Blue HER by T. beigelii (yeast)was studied at various temperatures (30–50°C), fasterdecolorization was observed at 37°C within 24 hincubation. Wang et al. (2009) studied decolorizationof Reactive Black 5 by a bacterial strain Enterobactersp. EC3. They found that, with an increase intemperature from 22°C to 37°C, the decolorizationrate increased and a further increase in temperature to42°C drastically affected decolorization activity of

Enterobacter sp. EC3. The optimum temperature fordecolorization was found to be 37°C. The authorsconcluded that the significant suppression of decolor-ization activity at 42°C might be due to the loss of cellviability or deactivation of the enzymes responsible fordecolorization at 42°C. Saratale et al. (2009a) studiedthe effect of temperature on decolorization of Scarlet Rby a microbial consortium consisting of two bacterialstrains, P. vulgaris and M. glutamicus. They found thatthe percent decolorization of Scarlet R at 30, 37, 40,45, and 50°C was 100, 100, 90, 82, and 45 after 24,14, 24, 36, and 48 h, respectively, by P. vulgaris while100, 100, 94, 70, and 50 after 36, 20, 30, 36, and 48 h,respectively, byM. glutamicus. These results show thatfavorable temperature range for decolorization by thesestrains was 30–37°C, with optimum temperature being37°C. All the above-mentioned studies indicate thatmicroorganisms degrade synthetic dyes best in therange of 25–37°C.

8.3 Initial Dye Concentration

The effect of initial dye concentration on microbialdecolorization of synthetic dyes is well studied. Thedecolorization of dye decreases with increasing dyeconcentration. The results of some studies showing theeffect of initial dye concentration on microbial decolor-ization of synthetic dyes are summarized in Table 6.

From Table 6, it is clear that, by increasing theinitial dye concentration, the decolorization is decreasedconsiderably. This decrease in decolorization withincrease in initial dye concentration is attributed to thetoxicity of the dyes to the growing microbial cells athigher dye concentrations. According to Gopinath et al.(2009), during the biodegradation of Congo Red by astrain of Bacillus sp., obtained from tannery industryeffluent, the increase in initial dye concentrationdecreased the decolorization rate, and at high concen-trations (1,500 and 2,000 mg L−1), inhibition wasobserved. According to Jirasripongpun et al. (2007),Enterobacter sp. was unable to grow in higher dyeconcentration, as it was dead when dye concentrationsof 50 and 100 mg L−1 of Reactive Red 195 were usedto test its decolorizing activity. The dye was consideredtoxic to the cells at high dye concentrations. Similarly,the results of Parshetti et al. (2006) have indicatedtoxicity of malachite green to Kocuria rosea MTCC1532 at higher dye concentration. Eichlerova et al.(2006) studied synthetic dye decolorization capacity of


Table 6 Effect of initial dye concentration on microbial decolorization of synthetic dyes

Microorganism Dyea (conc.) Decolorization (%) Incubation period References

Acremonium kiliense Malachite green (5 mg L−1) 95.4 72 h Youssef et al. (2008)(10 mg L−1) 35.48 NM

Citrobacter sp. Crystal violet (5 µM) 100 1 hb An et al. (2002)(2,000 µM) 55

Gentian violet (5 µM) 100

(2,000 µM) 42

Malachite green (5 µM) 100

(2,000 µM) 44

Brilliant green (5 µM) 100

(2,000 µM) 39

Basic fuchsine (5 µM) 98

(2000 µM) 27

Methyl red (5 µM) 100

(2,000 µM) 41

Kocuria rosea Malachite green (10 mg L−1) 100 2 h Parshetti et al. (2006)(30 mg L−1) 100 3 h

(50 mg L−1) 100 5 h

(70 mg L−1) 13 NM

(100 mg L−1) 6 NM

Lentinus polychrous Indigo carmine (10 mg L−1) 98.7 3 hb Sarnthima et al. (2009)(20 mg L−1) 97.6

(30 mg L−1) 96.5

(40 mg L−1) 93.8

Micrococcus glutamicus Scarlet R (50 mg L−1) 100 20 h Saratale et al. (2009a)(100 mg L−1) 100 28 h

(150 mg L−1) 100 36 h

(200 mg L−1) 38 72 h

(250 mg L−1) 18 72 h

Proteus vulgaris Scarlet R (50 mg L−1) 100 14 h Saratale et al. (2009a)(100 mg L−1) 100 24 h

(150 mg L−1) 100 30 h

(200 mg L−1) 50 72 h

(250 mg L−1) 25 72 h

Pseudomonas sp. SUK1 Reactive red 2(1,000 mg L−1)

95 18 h Kalyani et al. (2009)

(2,000 mg L−1) 91 48 h

(3,000 mg L−1) 88 72 h

(4,000 mg L−1) 84 NM

(5,000 mg L−1) 80 114 h

Trichosporon beigelii Navy blue HER (30 mg L−1) 100 12 h Saratale et al. (2009b)(50 mg L−1) 100 24 h

(70 mg L−1) 100 28 h

(80 mg L−1) 100 38 h

(100 mg L−1) 82 48 h

(150 mg L−1) 40 48 h

(200 mg L−1) 20 48 h

NM not mentioneda Dye used in a given study is the sameb Incubation period in a given study is the same unless otherwise indicated


a white-rot fungus Dichomitus squalens. They foundthat Malachite Green and Crystal Violet caused stronggrowth reduction even at small concentrations(50 mg L−1). Malachite Green at a concentration of100 mg L−1 inhibited the growth of D. squalenscompletely. Due to toxicity of the dyes to the microbialcells, the production of microbial biomass remainslower at higher dye concentrations. For example, themaximum cell growth yield of Aeromonas hydrophilawas about 1.2–1.6 gL−1 for dye concentrations of1,000–3,000 mg L−1 of Reactive Red 198, but it was0.7–1.0 gL−1 for dye concentrations of 4,000–8,000 mg L−1 (Chen et al. 2003). According to Yatomeet al. (1993), the decolorization of 2.3 µM crystal violetusing Nocardia corallina was complete upon contin-ued incubation for 24 h, but it decreased to <30% withincreasing initial concentration of crystal violet to4.5 µM. The growth of the cells was completelyinhibited at a dye concentration of 7 µM. According toYu and Wen (2005), both Pseudozyma rugulosa andCandida krusei could decolorize Reactive brilliant redK-2BP well at concentrations <200 mg L−1, decoloriza-tion reaching 99%. However, above this concentration,dye decolorization decreased sharply; at 1,000 mg L−1,only about 20% decolorization was achieved by thesestrains.

8.4 N Content in Medium

The supplementary sources of N in the growth anddecolorization media may affect the microbial decol-orization of synthetic dyes. The amount of nitrogenpresent in the media affects dye decolorization byaltering the enzyme production by fungi; for severalfungal species, the ligninolytic enzyme activity issuppressed rather than stimulated by high nutrient Nconcentrations (25–60 mM) (Kaushik and Malik 2009).This view is also supported by Hu (1998), who statesthat decolorization and mineralization of azo dyes havebeen reported to be enhanced in nitrogen-limited thanin nitrogen-sufficient cultures. Tatarko and Bumpus(1998) also reported that the addition of supplementalnitrogen only inhibited decolorization of Congo Red inplates containing high amounts of nutrient nitrogen.

8.5 Salts

Textile effluents contain various acids, alkalis, salts,or metal ions as impurities (Kaushik and Malik 2009).

Wastewaters from textile processing and dyestuffmanufacture industries contain substantial amountsof salts in addition to azo dye residues (Khalid et al.2008). Salt concentrations up to 15–20% have beenmeasured in wastewaters from dyestuff industries(EPA 1997). Thus, microbial species capable oftolerating salt stress will be beneficial for treatingsuch wastewaters. The identification of saline-tolerantbacteria that can degrade azo dyes may facilitate thedevelopment of biological treatment methods fortreatment of saline azo dye solutions using bioreactors(Khalid et al. 2008). Khalid et al. (2008) studied thepotential of Shewanella putrefaciens strain AS96 fordecolorizing four structurally different azo dyes(100 mg L−1) at different concentrations of NaCl.The results obtained showed that Reactive Black 5,Direct Red 81, Acid Red 88, and Disperse Orange 3were decolorized 100% in 8 h when NaCl concentra-tion was 0–40 gL−1. When NaCl concentration wasincreased to 60 gL−1, more time was needed fordecolorization, and in the case of Acid Red 88and Disperse Orange 3, percent decolorization alsodecreased significantly. Thus, at NaCl concentrationof 60 gL−1, Reactive Black 5, Acid Red 88, andDisperse Orange 3 were decolorized 100%, 53% and58%, respectively, in 24 h. Decolorization was notobserved at NaCl concentrations above 60 gL−1.

8.6 Shaking

There are contradictory reports about the effect ofshaking/agitation on microbial decolorization of syn-thetic dyes. According to some authors, decoloriza-tion is enhanced by shaking while according to othersby static conditions. According to Kaushik and Malik(2009), higher color removal is observed in shakingcultures because of better oxygen transfer and nutrientdistribution as compared to the stationary cultures. Onthe contrary, according to Kalyani et al. (2009),agitated culture of Pseudomonas sp. SUK1 showedalmost no decolorization in 24 h, while the staticculture decolorized more than 96% of the initial dyeconcentration (300 mg L−1) of Reactive Red 2 in 6 h.Similarly, Husseiny (2008), while studying the bio-degradation of Reactive Red 120 and Direct Red 81by Aspergillus niger, found that the static conditionswere more efficient than the shaking. Higher enzy-matic activities are observed in static conditions(Kaushik and Malik 2009). Novotny et al. (2004a)


studied the effect of liquid-medium culture type onthe synthesis of ligninolytic enzymes and the rate ofdecolorization of synthetic dyes by Irpex lacteus.They used Reactive Orange 16 and Remazol BrilliantBlue R at an initial concentration of 150 mg L−1 andmeasured the maximal enzyme activity (U L-1) ofMnP, laccase, and LiP and percent decolorization after7 days. They found that, in stationary culture, themaximal enzyme activity of MnP, laccase, and LiPwere 76±4.4, 2.0±0.0, and 1.1±0.2U L-1, respectively,while in submerged culture, these were 0.2±0.1, 1.0±0.1, and 0 UL-1, respectively. Similarly, the decolor-ization of Reactive Orange 16 and Remazol BrilliantBlue R in stationary culture was 85.8% and 99.7%,respectively, while in submerged culture, it was 16%and 97.8%, respectively. From these results, it isevident that maximal enzyme activity and decoloriza-tion were greater in stationary culture than in submergedculture. Saratale et al. (2009b) studied decolorizationand biodegradation of textile dye Navy Blue HER byT. beigelii NCIM-3326. They found that decolorizationof Navy Blue HER was 100% under static conditionand 30% under shaking condition. The growth of T.beigelii was also observed to be more under staticcondition (9.2 gL−1) as compared to shaking condition(4.2 gL−1).

8.7 Aerobic/Anaerobic Culture Conditions

It has been observed in a number of cases that theefficiency of aerobic treatment is inferior to that ofanaerobic decolorization process (Forgacs et al.2004). Under aerobic conditions, azo dyes are generallyresistant to attack by bacteria (Hu 1998). Bacteriausually degrade azo dyes under anaerobic conditionsto colorless toxic aromatic amines, of which some arereadily metabolized under aerobic conditions (Steffanet al. 2005). Except for a few, the aromatic aminesformed from decolorization of azo dyes are recal-citrant to biodegradation under anaerobic conditions(Pandey et al. 2007). Thus, although anaerobicreduction of azo dyes is generally more satisfactorythan aerobic degradation, the intermediate products(carcinogenic aromatic amines) have to be degradedby an aerobic process (Melgoza et al. 2004; Forgacset al. 2004) because the treatment of industrialeffluents containing aromatic compounds is neces-sary prior to their final discharge to the environment(Kalyani et al. 2009).

Lodato et al. (2007) studied the biodegradationof azo dye Acid Orange 7 by Pseudomonas sp. OX1under a variety of operating conditions. They foundthat growth and carbon source metabolism ofPseudomonas sp. OX1 were suppressed during theanaerobic stage, while the decolorization potential ofthe microorganism was fully exploited only in theabsence of oxygen. Azo dye Acid Orange 7 was nota growth substrate of Pseudomonas sp. OX1, norcould it be cleaved under aerobic conditions, whereas itwas effectively converted by the microorganism underanaerobic conditions. The authors suggested thatconflicting needs of providing both an aerobic environ-ment to promote growth/maintenance of the micro-organism and anaerobic conditions to favor cleavage ofthe azo bond and decolorization can be reconciled, inprinciple, by means of a cyclic process consisting ofalternating aerobic–anaerobic phases.

9 Decolorization of Real Dye Wastewaters

Real textile dye effluents contain not only dyes butalso salts, sometimes at very high ionic strength andextreme pH values, chelating agents, precursors,byproducts, surfactants, etc. (Wesenberg et al. 2003).Dyes of different structures are often used in thetextile processing industry, and therefore, the effluentsfrom the industry are markedly variable in composi-tion (Kalyani et al. 2009). The difficulties encoun-tered in the wastewater treatment resulting fromdyeing operations lies in the wide variability of thedyes used and in the excessive color of the effluents(Machado et al. 2006). Thus, in spite of the highdecolorization efficiency of some strains, decolorizing areal industrial effluent is quite troublesome (Wesenberget al. 2003). For opting biodegradation as the probableroute for treatment of wastewater, fungal strainscapable of growing in wide range of pH and tem-perature conditions and capable of resisting the toxicityof the dyes even at higher concentrations should bechosen (Kaushik and Malik 2009). Studies using realdye wastewaters in addition to pure and individual dyesolutions and simulated dye wastewaters should beconducted while evaluating the biodegradation capa-bilities of various microorganisms. Such studies will begreatly helpful in the feasibility and designing ofindustrial-scale bioreactors for treating dye waste-waters. Thus, researchers should not rely only on the


biodegradation studies of simulated dye wastewatersbut should extend their studies to the biodegradation ofreal dye wastewaters in a realistic approach.

10 Using Dyes as Sole Source of C and Energy

There are only very few bacteria that are able to growon azo compounds as the sole carbon source; thesebacteria cleave –N=N– bonds reductively and utilizeamines as the source of carbon and energy for theirgrowth, but such organisms are specific towards theirsubstrate (Pandey et al. 2007). Ren et al. (2006),while studying the decolorization of triphenylmeth-ane, azo, and anthraquinone dyes by a newly isolatedA. hydrophila strain, found that crystal violet(50 mg L−1) could be used as sole carbon and energysource for cell growth. In our own laboratory, whenMalachite Green was used as the sole source of C inthe decolorization medium, two fungal species,Aspergillus flavus and Alternaria solani decolorizeda 30-µM solution by 99.78% and 91.72% respectivelywithin 6 days (Ali et al. 2009). This showed that boththe species were able to use the dye for their growth.Saratale et al. (2009b), while studying the decoloriza-tion and biodegradation of a textile dye Navy BlueHER by T. beigelii NCIM-3326, found that this yeastcan utilize the dye and its reaction intermediates as acarbon source, achieving a nearly complete mineral-ization of the dye compound (95% TOC removal after24 h). Microbial strains capable of utilizing the dye assole source of C and energy for their growth are ofspecial interest. Such microorganisms eliminate thepollutant in a real sense. These microorganisms mayconvert the undesirable chemicals (pollutants) intouseful products like organic acids, alcohols, etc. Muchattention should be focused on such microbes becausesuch microbes may prove best candidates for greensynthesis in future.

11 Characterization of Biodegradation Products

The study of the products of biodegradation ofsynthetic dyes is important in order to know aboutthe environmental fate of these pollutants. It is veryimportant to analyze the treated water with regard tothe dye content as well as intermediates, especiallyaromatic amines, since some are considered carcino-

genic (Forss and Welander 2009). The removal ofcolor from dye-containing wastewater may be the firstand a major concern (Sun et al. 2009), but the aim ofbiodegradation of dyes is not only to remove the colorbut also to eliminate or substantially decrease thetoxicity of the dyes (detoxification). Detailed charac-terization of the intermediates and metabolites pro-duced during biodegradation must be done to ensurethe safety of the decolorized wastewater (Kaushikand Malik 2009; Couto 2009). Various basic andadvanced instrumental techniques of chromatographyand spectroscopy can be used to isolate and charac-terize the products of biodegradation of dyes and thushave an insight into themechanism of biodegradation. Todate, very few reports are available on the intermediatesor the products of biodegradation of triphenylmethanedyes (Chen et al. 2008). The bacterial metabolism of azodyes is initiated in most cases by a reductive cleavageof the azo bond, which results in the formation ofcolorless aromatic amines (Khalid et al. 2008).

Relatively simple techniques of UV-visible spec-trophotometry and thin layer chromatography (TLC)can be used to know whether decolorization hasoccurred through adsorption of dye particles on themicrobial cell surface or through breakdown of thedye structure by the living microbial systems or both.In adsorption, examination of the absorption spectrumwill reveal that all peaks decrease approximately inproportion to each other, but if the dye removal isattributed to biodegradation, either the major visiblelight absorbance peak will completely disappear or anew peak will appear (Chen et al. 2003; Saratale et al.2009b). In addition, in adsorption, cells may becomedeeply colored because of adsorbing dyes, whereasthose retaining their original colors are accompaniedby the occurrence of biodegradation (Sun et al. 2009).Similarly, by comparing the Rf values of the biodeg-radation products with those of the original dye andknown standards on TLC, some useful informationcan be obtained about the nature of the biodegrada-tion products. More advanced techniques like gaschromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC) andnuclear magnetic resonance (NMR) spectroscopy canbe used to get more authentic and detailed informationabout the products of biodegradation and also about thestepwise mechanism of biodegradation.

Quantification of CO2 and NH3 produced in theincubated culture medium can also provide some


information about the mechanism of decolorization.Chen et al. (2008) studied the biodegradation of crystalviolet by a Shewanella sp. NTOU1 and quantified CO2

and NH3 produced in the medium after color removal.According to them, after color removal, nearly 45% ofcarbon and 39% of nitrogen in the crystal violet wereconverted to CO2 and NH3, respectively. Furthermore,after 48 more hours of incubation after color removal,70% of the carbon and 61% of the nitrogen in thecrystal violet were converted to CO2 and NH3,respectively. Based on these data, they suggested thatdecolorization of crystal violet by this strain was aresult of biodegradation.

12 Mechanisms and Pathways of Biodegradationof Dyes

Detailed characterization of biodegradation productsof synthetic dyes isolated at different stages of theprocess can help in proposing and establishingmechanisms and pathways by which microorganismsdegrade dyes. Different microorganisms may havedifferent pathways for degrading different dyesdepending upon the dye structure, strategy of themicrobial system for dye degradation, and many otherfactors. Even small structural differences can affectthe decolorization process (Eichlerova et al. 2006). Amajor mechanism behind biodegradation of syntheticdyes in microbial systems is because of the biotrans-formation enzymes (Raghukumar et al. 1996). It isgenerally recognized that azoreductases play animportant role in bacterial dye decolorization (Kuhadet al. 2004). Among synthetic dyes, azo dyes constitutethe largest and most versatile class with the greatestvariety of colors (Bafana et al. 2009). The mechanismof microbial degradation of azo dyes involves thereductive cleavage of azo bonds (–N=N–) with the helpof azoreductase under anaerobic conditions resultinginto the formation of colorless aromatic amines (Changet al. 2000; Chen 2006; Saratale et al. 2009a).

Only a limited number of studies have attemptedmolecular characterization of dye decolorization(Kuhad et al. 2004). There is still a gap in the degrada-tion mechanisms of dyes by white-rot fungi and theirligninolytic enzymes (Couto 2009). The chemistry ofbiodegradation of synthetic dyes and other organicxenobiotics is an interesting and emerging researcharea. With the help of advanced instrumental analytical

techniques, it is possible to study the various inter-mediates and final products of microbial degradationof synthetic dyes in detail. Such studies are of highvalue in many ways. Recently, some researchers havestudied the mechanisms and pathways of biodegrada-tion of dyes. Here, some of these studies are discussedin which the proposed mechanisms and pathways ofbiodegradation of Direct Black 38 (azo dye), CottonBlue (triphenylmethane dye), and Reactive Blue 5(anthraquinone dye) have been presented.

Bafana et al. (2009) studied decolorization anddegradation of a benzidine-based azo dye, DirectBlack 38 by Enterococcus gallinarum. Benzidine and4-Aminobiphenyl were identified by HPLC-MS asthe degradation products. The mechanism of biodeg-radation of this dye by Enterococcus gallinarum isgiven in Fig. 1. Shedbalkar et al. (2008) studiedbiodegradation of triphenylmethane dye Cotton Blueby P. ochrochloron MTCC 517. The GC-MS analysisof extracted metabolites showed the formation ofsulfonamide (MW 172, mass peak at 172 m/z) andtriphenylmethane (MW 343, mass peak at 343 m/z).The mechanism of biodegradation of this dye byP. ochrochloron is given in Fig. 2. Sugano et al.(2009) studied degradation pathway of a commercialanthraquinone dye, Reactive Blue 5 catalyzed by a dye-decolorizing peroxidase, DyP from Thanatephoruscucumeris Dec 1. The products obtained were inves-tigated using electrospray ionization mass spectrometry(ESI-MS), TLC and 1H- and 13C-NMR. Their deducedmechanism for biodegradation of Reactive blue 5 isshown in Fig. 3.

13 Phytotoxicity and Microbial Toxicity of Dyesand their Biodegradation Products

It is very important to know whether biodegradationof a dye leads to detoxification of the dye or not. Thiscan be done by performing phytotoxicity and micro-bial toxicity tests of the original dye and itsbiodegradation products. In phytotoxicity studies,the seeds of model plants can be treated with aparticular concentration of the original dye and alsowith its biodegradation products. The effect of thetreatment on percent germination and length ofplumule and radicle can be evaluated and the resultscompared with those of the control (without treatmentwith dye and its biodegradation products). Differences


in results can be used to know whether the degrada-tion products are less toxic to the growing plants thanthe original dye or not. The same procedure can alsobe used for microbial cultures, and the antimicrobialactivity (toxicity) of the original dye and its degradationproducts can be compared. In this case, the number ofmicrobial cells per milliliter will be counted. Suchstudies can also be conducted using algae, in whichcase, “chlorophyll a” content in cultures treated with theoriginal dye and in those treated with its degradationproducts can be compared.

Kalyani et al. (2009) conducted phytotoxicitystudy of Reactive Red 2 and its degradation productsusing Sorghum vulgare and Phaseolus mungo asmodel plants. They treated the plant seeds with water,Reactive Red 2 (5,000 ppm), and its extractedmetabolite (5,000 ppm) separately and comparedpercent germination and the lengths of plumule andradicle. From the results, it was found that themetabolites produced after the biodegradation ofReactive Red 2 were less toxic compared to theoriginal dye. Similarly, Parshetti et al. (2006) studiedthe phytotoxicity of Malachite Green and its degra-dation product using Triticum aestivum and P. mungoas model plants. They found that percent germinationof both T. aestivum and P. mungo seeds was less withMalachite Green treatment as compared to its degra-

SO3

N

NH

SO3

N

H

SO3

Cotton blueM.W. 799.82

Lignin peroxidaseAsymmetric cleavage

+

NH2

SO3

Triphenylmethane SulfonamideM.W. 343 M.W. 172Mass peak (m/z) 343 Mass peak (m/z) 172

Fig. 2 Proposed pathway for the degradation of Cotton Blueby Penicillium ochrochloron MTCC 517 (adapted fromShedbalkar et al. 2008)

N N

S O Na- +

O

O

S

N

HO

H2NNN

NH2N

NH2

O

O

ONa -+

Direct black 38

NH2H2N

Cleavage of azo bonds

Benzidine

H2N

Deamination

4-Aminobiphenyl

Fig. 1 Scheme of degrada-tion of Direct Black 38 byEnterococcus gallinarum(adapted from Bafanaet al. 2009)


dation product and distilled water. The MalachiteGreen significantly affected the length of plumule andradicle than its degradation product. They concludedthat the degradation product was less toxic. Dawkar etal. (2009) carried out phytotoxicity analysis of textileazo dye Navy blue 2GL and its metabolites formedafter degradation by Bacillus sp. VUS. UntreatedNavy Blue 2GL (5,000 ppm) showed 80% and 70%germination inhibition in Sorghum bicolor andT. aestivum, respectively, after 7 days of incubation.There was no germination inhibition in both the seedswhen metabolites formed after complete decolorizationwere applied at the same concentration. Reduction(2–5%) in the growth (shoot and root lengths) wasobserved in the presence of metabolites when comparedto the growth in distilled water in both the plants.

Jadhav et al. (2008) studied phytotoxicity of Methyl redand its metabolites formed after biodegradation byG. geotrichum MTCC 1360. The untreated dye at300 mg L−1 concentration showed 88% germinationinhibition in S. bicolor, whereas it was 72% in T.aestivum. There was no germination inhibition for boththe plants by Methyl Red metabolites at 300 mg L−1

concentration. Saratale et al. (2009a) conducted phy-totoxicity studies of Scarlet R and its metabolitesformed after biodegradation by a mixed microbialculture, consortium-GR (consisting of bacterial strainsP. vulgaris and M. glutamicus). They used the dye andits extracted metabolites at a concentration of3,000 ppm for the germination and growth of twoplants, P. mungo and S. vulgare. The results showedthat the dye caused a germination inhibition of 50%

O

O

NH2SO3H

NHHN

NSO3H

N

N

Cl

HN SO3H

Reactive blue 5MW: 773.5

COOH

COOH

Product 1Phthalic acidMW: 166

+

NH2SO3H

HNHN

NSO3H

N

N

HN SO3H

Cl

NH2SO3H H2N

+

HN

SO3HN

N

N

Cl

HN SO3H

Product 2MW: 472.5

SO3H

N N

SO3H

Product 42,2'-Disulfonyl azobenzeneMW: 342Corresponding dimer and/or polymer

H2N

SO3H

+

H2N

N

N

N

HN

HO3S

Cland or

SO3HH2N

+

H2NHN

SO3HN

N

N

ClProduct 3MW: 301.5

Fig. 3 Deduced degrada-tion pathway of ReactiveBlue 5 treated with a uniqueperoxidase DyP fromThanatephorus cucumerisDec 1. Compounds shownin square brackets were notdetected by ESI-MS(adapted from Suganoet al. 2009)


and 60% in P. mungo and S. vulgare, respectively. Theextracted metabolites caused no germination inhibition.The length of radicle and plumule was also signifi-cantly more in case of extracted metabolites ascompared to that in the dye. Similarly, Saratale et al.(2009b) carried out phytotoxicity studies of Navy BlueHER and its metabolites produced after completedecolorization by T. beigelii NCIM-3326. They usedthe dye and its extracted metabolites at a concentrationof 1,500 ppm for the germination and growth ofP. mungo and S. vulgare. The phytotoxicity studiesshowed good germination rate as well as significantgrowth in the plumule and radicle for both the plants inthe metabolites extracted after decolorization as com-pared to the dye sample. Thus, Navy Blue HER wasdetoxified by T. beigelii. Shedbalkar et al. (2008)compared phytotoxicity of Cotton Blue and itsextracted metabolites formed after biodegradation byP. ochrochloron MTCC 517. They used Cotton Blueand its extracted metabolites at a concentration of700 ppm for the germination and growth of T. aestivumand Ervum lens L. The dye caused 30% and 20%germination inhibition in T. aestivum and Ervum lensL., respectively, whereas the extracted metabolitescaused only 10% germination inhibition in T. aestivumand no germination inhibition in Ervum lens L. Thelength of shoot and root was also significantly more incase of extracted metabolites than in the dye. The sameauthors also conducted a microbial toxicity test of thedye and its biodegradation products. The microbialtoxicity on Azobacter vinelandii, a nitrogen fixingbacterium, showed growth inhibitory zone (1.2 cm)surrounding the well containing dye, while theproducts did not show inhibitory zone.

Chen et al. (2008) performed antimicrobial test ofcrystal violet and its degradation product using E. colistrain JM 109 as model microbe. They used crystalviolet solution (100 mg L−1) before biodegradation,crystal violet solution after incubation (with Shewanelladecolorationis NTOU1) for 11 h (>98% decolorized)and crystal violet solution after incubation for 59 h,for growth of Escherichia coli strain JM 109. Theycounted cell number (cells mL−1) of the E. coli strainJM 109 in the test tubes after incubation with crystalviolet or its degradation products for 1, 12, and 24 h.The data showed that crystal violet solution wastoxic but the crystal violet solution after incubationwith the S. decolorationis NTOU1 for 11 h or 59 hwas not toxic to the E. coli strain JM 109. They

concluded that S. decolorationis NTOU1 coulddetoxify crystal violet during decolorization process.Parikh and Madamwar (2005) cultured cyanobacteria,Gloeocapsa pleurocapsoides, and Phormidiumceylanicum in 100 mg L−1 FF Sky Blue andChroococcus minutus in 100 mg L−1 Amido Black10B and measured their “chlorophyll a” content(mg L−1) after 26 days of incubation. The resultsshowed a large decrease in “chlorophyll a” contentoccurred in presence of the dyes. They concluded thatthis was due to attenuation of light transmission by thedyes, which then reduces the rate of photosynthesisand ultimately the growth rate.

14 Future Perspectives

Biodegradation of synthetic dyes using differentfungi, bacteria, yeasts, and algae is becoming apromising approach for the treatment of dye waste-waters. Green plants are nature’s factories, which fixinorganic chemicals (CO2 and H2O) into organic form(glucose and then other complex molecules) throughphotosynthesis and other reactions, while microbesare the nature’s tools, which convert back the organicmaterials (dead bodies of plants and animals) toinorganic form (CO2, H2O, and salts) throughdecomposition and mineralization. Thus, green plantsand microbes are responsible for keeping a balancebetween the organic and inorganic worlds. With theincreasing production of synthetic chemicals and theirultimate release into the environment, the naturalmicrobial populations are unable to decompose themin due course of time. As a result, such chemicals areaccumulated in the ecosystem and affect the quality oflife. Keeping in mind the increasing production ofthese chemicals and their persistence in the naturalenvironment, their removal is utmost necessary. Byexploiting the biodegradation potentials of differentmicrobes, it is possible to handle the problem in abetter way. A better understanding of biodegradationof synthetic dyes requires knowledge of chemistryand microbiology, whereas its application on indus-trial scale requires knowledge of biochemical engi-neering as well. Thus, research in this field is highlyinterdisciplinary in nature. Now, since interdisciplin-ary research is highly encouraged and valued world-wide especially in broad minded communities, it isfully hoped that the science and technology of


biodegradation of organic xenobiotics will emerge asa leading one for control of environmental pollution.An understanding and knowledge of biodegradationare not only helpful in pollution abatement but alsoin the production of biofriendly and environmentfriendly products like biodiesel, bioethanol, biopesti-cides, biopolymers, etc.

The biodegradation abilities of microorganisms canbe enhanced by gradually exposing them to higher con-centrations of synthetic organic chemicals. Adaptationof a microbial community toward toxic or recalcitrantcompounds is found to be very useful in improving therate of decolorization process (Dafale et al. 2008). Theadaptation of microorganisms to higher concentrationsof pollutants is called acclimatization and leads toforced or directed evolution. Microorganisms exposedto higher levels of pollutants evolve mechanisms andpathways for handling (degrading) them. This happensthrough expression of genes encoding for enzymesresponsible for degradation. Alternatively, identifica-tion, isolation, and transfer of genes encoding fordegradative enzymes can greatly help in designingmicrobes with enhanced degradation capabilities.Thus, acclimatization and genetic engineering bothcan be helpful in designing super-degraders. Out of thetwo approaches, acclimatization is natural, since in thiscase, the built-in genetic setup of the microorganism isnot disturbed; only some components are enabled. Onthe other hand, in genetic engineering, the naturalgenetic setup of the microorganism is changed byincorporating new gene(s). Therefore, many scientists(especially environmentalists) are skeptic about theusefulness of genetically modified organisms. Theyfear that such modified organisms will create newenvironmental problems. Time will prove or disprovethe reality of such fears.

15 Conclusions

Synthetic dyes released into the environment causeconsiderable water and soil pollution because theymay be toxic, carcinogenic, mutagenic, and clasto-genic to living organisms. Over the last two decades,awareness and concern about the environmental andhealth hazards of synthetic dyes is increasing in theglobal community. Consequently, environmental andgovernment legislations are becoming more and more

tight regarding the removal of these pollutants fromindustrial wastewaters. Different physical and chemicalmethods have been employed for the treatment ofsynthetic dyes wastewaters. These methods mostlysuffer from serious limitations, like high cost, lowefficiency, limited versatility, and production of second-ary pollution (sludge), etc. In contrast, bioremediation isa cost-effective, efficient, biofriendly, and environmen-tally benign method for removal of dyes from industrialwastewaters. Bioremediation is the application ofmicroorganisms (fungi, bacteria, actinomycetes, yeasts,and algae) for the removal of xenobiotics (syntheticorganic compounds, which are not found in nature andare thus foreign and new to the biota) from pollutedenvironments. Microorganisms are our microscopicallies, which can help us clean the contaminated soilsand waters. They are tiny biological reactors, whichcan convert harmful synthetic chemicals (organicpollutants) into simple, less toxic, or completely benignproducts. Microorganisms can even mineralize organicpollutants, that is, to completely degrade them andconvert into water, carbon dioxide, and salts. Thebiodegradation of synthetic dyes is affected by manyfactors like pH, temperature, dye concentration, nitrogencontent in culture medium, presence of salts, agitation,aeration, etc. Therefore, these factors are to be taken intoaccount while evaluating the biodegradation abilities ofdifferent microorganisms. Microorganisms capable ofusing the dye molecules as a sole source of carbon,nitrogen, and energy are of special interest and signifi-cance because they consume the dye for their growth andactivities while at the same time eliminate the pollutant ina real sense. Such microorganisms are a valuable giftfrom Nature. Their biodegradative potentials can beexploited to deal with the problem of synthetic dyes’pollution and explore new horizons for further research.Over the past two decades, much attention has beenfocused on the biodegradation of synthetic dyes usingdifferent groups of microbes. The science of bioremedi-ation is emerging as a unique tool to deal with theremoval of synthetic dyes and other xenobiotics from theenvironment.

Acknowledgements I am grateful to Dr. Muhammad Ali,Assistant Professor of Analytical Chemistry and Chairman,Department of Biotechnology, University of Malakand, for theencouragement and to my student Mr. Shah Khalid Muhammadfor the help in establishing the environmental biotechnologyresearch section at the department.


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