.Indian Journal of Experimental Biology Vol. 4 1, September 2003, pp. 1046- 1067

Anaerobic biodegradation of aromatic compounds

P lothimani , G Kalaichelvan, A Bhaskaran, D Augustine Selvaseelan & K Ramasamy*

Fermentati on Laboratory, Tamil Nadu Agricultural Uni versity, Coimbatore 64 1003. India

M any aromatic compounds and their monomers arc ex isting in nature. Besides they are introduced in lo the environment by human acti vity. The conversion of these aromatic compounds is mainl y an aerobic process because of Ihe in vol vement of molecular oxygen in ring fi ss ion and as an electron acceptor. Recent literatures indica ted that ring fi ss ion of monomers and obligomers mainl y occurs in anaerobic environments through anaerobic respirati on w ith nitrate, sul phate. carbon dioxide or carbonate as electron acceptors. These anaerobic processes wi ll help to work out the better situati on for bioremed iation of contami nated envi ronments. While there are plenty of efforts to reduce the release of these chemica ls to the environment , already contaminated sites need to be remediated not onl y to restore the sit es but to prevent the leachates spreading to nearby environment. Basicall y microorganisms are better cand idates for breakdown of these compounds because of their wider cataly tic mechanisms and the ability to act even in the absence of oxygen. T hese microbes can be grouped based on their energy mechanisms. Normall y , the aerobic counterparts employ the enzymes like mono-and-di -oxygenases . The end product is basically catechol, which further may be metabolised to CO2 by means of quinones reduc tascs cycles. In the absense of reductases compou nds, the reduced ca techols tend to become ox idised to form many quinone compounds. The quinone products are more reca lcitrant and lead to other aesthetic problems li ke colour in water, unpleasant odour, etc. On the contrary , in the reducing environment thi s process is prevented and in a cascade of pathways. the cleaved products arc converted to acetyl co-A to be integrated into other ceniral metabolite paths.

The central metabolite of anaerobic degradati on is invariably co-A thi o-esters of benzoic acid or hyd roxy benzoic acid. The benzene ring undergoes vari ous subst ilUtion and addi ti on reacti ons to form chloro' , nitro' , methyl' compounds. For complete degradation the side chains musl be removed first and then the benzene ring is acti va ted by ca rboxy lation or hydroxy lati on or co-A thioester format ion. In the next step the activated ring is converted to a form th at can be co llecled in the central pool of metaboli sm. The third step is the channel ing reaction in which the producl s of the cata lysis arc di rected into cen tral metabolite pool. The enzymes invo lved in these mechanisms are mostl y benzy l co-A ligase. benzy l alcohol dehydrogenase. Other enzy mes involved in thi s path are yet to be purified though many o f the react ions products that have been theoreti ca lly postul ated have been identified. Thi s is mainly due to the instability of intermedi ate compounds as well as the association of the enzy me substrate is femoral and experimental cond itions need to be sophi sticated further for iso lat ion of these enzymes . The first structural genes of benzoate and hydroxy benzoate li gases were isolated from Rhor/opseudolllollas pallisl/·is. This gene clus ter of 30 kb size fou nd in Rhodopseudolllollas pailis/ris coded for the Bad A protein. Simil arl y , some of the bph A.B ,C and D cluster of genes coding for the degradation of pentachlorobenzenes were located in Pselidolllonas pselidoaigaligell esKF 707.

Keywords: A naerobic biodegradation , Aromatic pollutant. Biodegradation, Bioremed iation

A large variety of aromati c substances participate in our life processes and their biosynthesis and degradation form an integral part of the natural carbon cycle. Human ac ti vities are additional sources of synthetic organic chemical s in the environment The persistence of anthropogenic aromatics 111 the environment has caused great concern due to their tox icity, mutagenicity and bioconcentration in higher organisms. These aromatic compounds belong to the most second abundant family of organic consti tuents present in the biosphere. Due to the industrial revolution , a wide variety of aromatic compounds have been introduced into the environment. Aromatic

*Correspondent author : E-mai l : ramasamy@yahoo.eom; Fax : 0091-422-2431672

hydrocarbons and their derivati ves are used tI1

increasing amounts tI1 a number of industrial operations such as the manufacture of chemi cal solvents, pesticide production and other petroleum base industries, Consequently there is also a concomitant increase in the di sseminati on of these material s into the environment: for exa mple, the deliberate application of pesticides in fields for pest control, acc idental spillage of chemicals during any phase of their use, seepage of chemicals fro m di sposal areas into natural waters,

Though government regul atory agencies and market processes, industries are slowly modifying their processes to decrease aromatic wastes. Complete prevention of aromatic pollution has been proposed as a long term goal; there is still a great need to

.,

JOTHIMANI et al.: ANAEROBIC BIODEGRATION OF AROM ATIC COMPOUNDS 1047

remediate the present di scharges and aro matic pollution inheri ted fro m the past. The use of microorganisms to degrade aromatic po llutio n is an interesting low cost approach fo r the treatment of industrial effl uents, contaminated sediments, so il and ground water. The vast number of studies in this fie ld parti cularl y in the degradatio n of li gnin by white rot fungi that resulted in the complete understanding of the metaboli sm, enzymes and genes involved 1.2.

The introduction of syntheti c chemicals into the environment, whether de liberate or acc idental, is not merely a concern but imposes o n us a responsi bility to fully explore the limitatio ns and characteri sti cs of the biological processes on which the ir decomposition depends. If biodegradatio n does not occur in the wate r column or in aerobic so il s, these compounds may eventually end up in anaerobic sinks and sediments. The kinds of anaerobic environments in which they may eventuall y reside, and the microbi all y mediated transformations known to occur in the absence of molecular oxygen like wise- the different aspects of aromatic compounds and its degradation in anaerobic systems are di scussed in thi s rev iew.

Types of aromatic compounds , Aromatic hydrocarbons

Polynuclear aromatic hydrocarbons (HC) are formed due to fusion of benzyl rings. Polycyclic aromatic HC includes naphthalene(C ,oHs), acenaphthalene (C I2Hg),

fl urocene(C 13H1o) and phenantherene(C I4H 1o). Thi s aromati c hydrocarbo n, such as a lky lbenzenes

(2), the ary lmethanes (3), b iphenyl (4) and condensed polycyclic systems (5) and anthracene (6) had exhibited the chemi cal reacti vity and spectroscopic features.

Aromatic nitrogroups The importance of aro matic nitrogroups ari ses

paI1icuiarl y from the ready conversion reaction of the nitrogroups into other functio nal groups, princ ipally by routes in volving initi a l red uctio n to the amino group.

Aromatic halogen compounds These aromati c compounds are those in

which the ha logen is directl y attached to the aromatic nucleus (e .g. C6HsC I or PhCl , (chl orobenzene) and those in which the halogen is substituted into an a lky l side chain (e.g. C fiHs-C H2Cl or Ph-CH2C l(benzy l chloride).

Aromatic sulphonic acids and their derivatives The fo llowing structures indicated the range of

aromatic sulpho nic ac ids (l), sulphinic acids(2), sulphony l chlorides(3), sulpho namides(4) and sulphonate esters(S) .

Aromatic amines Primary amines, secondary amines and terti ary

amines where the amino group is attached to the aromatic ring at diffe rent pos itions.

Substitution products of aromatic amines Nuclear substitutio n products. Acety lated amines and substitutio n products.

Diazonium salts Primary aromatic amines o n reaction with nitrous

ac id in the presence of hydrochlori c and at about O°C yie ld di azonium salts as di sc rete intermedi ates.

Miscellaneous aromatic nitrogen compounds - ego Azoxybenzene

Aromatic aldehydes - fo rmed by modi fi cation of aromati c ring substitutions.

Aromatic ketones and quinines

Aromatic carboxylic acids

Sources of aromatic compounds and causes Important natural sources of aromatic compounds

are in genera l of poorl y biodegradable po lymers such as li gnin , condensed tannins and humus. Other aromatic compounds of lower mo lecul ar weight such as fl avonoides, aro matic amino ac ids and pheno lic ac ids account fo r the bul k of more readi ly metabo lizable aromatic compounds occurring in the natura l enviro nment\ Since fro m the start of the industri al revolutio n a wide vari e ty of syntheti c aromatic compounds have a lso been introduced in the enviro nment thro ugh anthropogenic activi ty . Such sources of the anthropogenic aro rnati cs4 are given in T ables 1,2.

Aromati c compounds o f most concerned are those, which are industri ally produced . With inc reased producti on there is inc reased loss in to the environment during a ll phases of use. From 1940 to 1970, the production of syntheti c organic chemical rose fro m 2.5 mt to abo ut 90 mt. It has been estimated that nearl y 200 thousand new organic compounds are sy nthes ised every yea r in the world , and that over 1000 will eventuall y be put to some use.

1048 INDI AN J EX P BIOL, SEPTEMB ER 2003

Coal and petro leum are the starting materi als for the syn thes is of most organic chemicals, and fro m the mid 1800' s they have been the bas is fo r the fa 1'­

reaching petrochemical industri es. Most of the aromatic hydrocarbons supply (more th an 70%) comes from petro leum with the remainder be ing deri ved fro m coal and imports. When bituminous coal

is heated at 100o-1300°F in the absence of a ir, a li ght o i I frac tion is separated fro m the tar. The latter suppli es the po lycyclic aromatic hydrocarbons naphthalenes and anthracenes while the light o il suppli es benzene, to luene, xy lenes and solvent naphtha's. Preparation of aro mati c hyd rocarbons fro m crude oil requires d isti llation, so lvent ex traction and crysta lli zati on process. The uses of these compo unds in industry are numerous. They are the starting materi a ls for the sy nthes is of pl astics, pa ints , pesti c ides, res in s and dyes. They are a lso used as solvents for paints, dyes, res ins, rubber and plasti cs , and are used in av iatio n and auto moti ve fue ls. Benzene, for example, is ex treme ly versatil e and used for the sy nthesis of plasti cs (eg. styrenes), pesti c ides (chloro benzenes), dyes (anilene), solvent (aceto ne), adhes ives (resorc ino l) and res ins(phenol).

Halogenated aromatic compounds such as 2,4,5 tri chloro phenoxy aceti c ac id (2,4,5 T ), d ichloro d iphenyl trichloro ethane (DDT), po lychlorinated and po ly brominated bipheny ls (PC B and PBBs), and

chlorinated di ox ins have been well pub li sed in te rms of the ir delete rious effec ts on both human health and on the environment. Acute health effects inc lude sk in les ions, abnormal lever func tio ns, neuro logical effect such as numbness and visio n impa irment , suppress ion of the immune syste m and interfe rence with reproducti ve hal·mones . Ev idence is also mounti ng with regard to the ir carc inogenic and teratogenic effects. Damaging effects o n the enviro nment inc lude reduced photosynthetic acti vity and loss in spec ies di vers ity in phytoplankton popul atio n. Because of the bi omagni ficati on of ha logenated hydroca rbons th rough the food chain , high concentratio ns ha e accumul ated in popul ations of bird species and have interfered with the ir reproducti ve capac ity. T hi s subsequently caused a precipito us decline in the popul atio n of certa in species. Sea ls and sea lion populations were reported to be affected in a sim ilar manner since it was banned fro m w ide spread use, a dec line in the levels of DDT in certa in ani ma l popul ati ons has been observed. Thi s is also refl ec ted in a decline in the genera l level fo und in the hu man tissue. The mean concentratio n in human fa t ti ssue was 7.88 ppm in 1970 , and had dropped to 4.99 ppm in 1974.

Entry of PC Bs, PBBs, d ioxins and nonchl orinated aromatic hrdro carbons into the envi ronment appears to be mainly th rough disposal in land fill s and is

Table I - Aromatic com pounds o f anth ropogenic act ivity

Aromatic compou nds

BTEX* Styrene PAH* Alkyl phenols Aromatic sulfonates Aromatic amines Azo aromati cs Nit ro-aromatics Chl orophenols and diox ins Chloro aromat ic hydro carbon and PCB*

Industria l sources

Fossil fue ls, solvents, ind ustri al feed stocks Plast ics Fossil fue ls, wood preservation Surfac tants, detergents Surfac tants, detergents, sulfite pulping, dyes Pes ticides, dyes, pigments, pharmaceuticals Dyes Explosives, pharmaceuticals, pesticides, dyes Wood preservation, pes ticides, pulp bleaching effl uents Pestic ides, solvents, dielec tric and hydraul ic flu ids

BTEX: PAH PCB:

Benzene, toluene, e thyl benzene and xylene Polycyclic aromatic hydrocarbons Poly chlorinated biphenyl

Compound

Lignin

Table 2 - Aromatic compounds o f natural orig in

Examples

Aromatic amino ac ids Quinones

Coni fery l alcohol and other li gnin precursors Tyrosine, tryptophan Pl astoquinone

Phe no lics Tannins Others

Phenols, phenolic acids, coumari ns. ci nnamic acid etc. Gallotannins, vegetable tannin deri vati ves Melanins, flavonoids, hormones, alka loids and terpenes.

JOTHIMANI et al.: ANAEROBIC BIODEGRATION OF AROMATIC COMPOUNDS 1049

sewage treatment facilities, evaporative loss into the atmosphere, acidents such as leaks of heat exchanger fluid and industrial fires, fly ash and flue gases from municipal incinerators, agricultural and urban run off, and discharges from industry and domestic treatment plants after processing.

Originally the aromatic pollutants, such as halogenated aromatics and nitroaromatics are foreign to the environment and consequently these have been termed as "Xenobiotic Compounds". The persistence of the anthropogenic aromatics in the environment has caused a great concern due to their toxicity , mutagenicity and bioconcentration In higher organisms. Complete degradation of aromatic compounds to gaseous metabolites is preferred to prevent the accumulation of toxic end products. Benzoic acid is one such metabolite that is hydroxylated to 2 hydroxy benzoic acid (Salicylic acid) under anaerobic decomposition. Benzoic acid is highly toxic to crop plant and salicylic acid decreases the uptake of nutrients like nitrogen, phosphorus and potassium. It also reduces the nitrate reductase activity of crop plants5

.

Microbial degradation and metabolic fate of aromatic compounds

Anaerobic or aerobic biodegradation capacities can be selected depending on whether molecular oxygen is estimated or supplied . In the biosphere, there is approximately, a 40,000 times excess of molecular oxygen compared to organic carbon indicating that most organic matter degradation occurs in aerobic environments. However, if organic pollution occurs in saturated environments, anaerobic degradation will most likely predominate due to the low solubility and slow mass transport of O2 in water. The biosphere harbours host of different habitats within which microorganisms can operate, provided nutrients are available and physical conditions are appropriate for mineralization to occur.

Under aerobic conditions molecular oxygen is required not only as the terminal electron acceptor during respiration, also for inserting the compounds during hydroxylation and ring cleavage. Oxygenase enzymes mediate these reactions. In the absence of oxygen, microorgani sms necessarily have evolved different mechanisms for ring fission. The earliest evidence for anaerobic catabolism of the aromatic nucleus was suggested by Boruff and Buswe1l6 ,who examined the anaerobic fermentation of lignin-upto 54% of the lignin was converted to CO2 and CH4.

Since lignin is a highly aromatic polymer, loss of such fraction of the lignin would likely have invol ved fermentation of aromatic constituents. More specific evidence was reported later by Tarvin and Buswe1l 7

,

who demonstrated the anaerobic metabolism of benzoate, phenyl acetate , hydro cinnamate and cinnamate. complete decomposition of these compounds was observed and all of the carbon was accounted for as CO2, CH4 and cells.

Aerobic degradation and pathways of aromatic ring cleavage

The microbial degradation of aromatic compounds are done by ring fission which is predominantly an aerobic process. The microorgani sms degrade these compounds either totally or partially depending on the number of aromatic units and especially on the type of substituents. The basic reaction sequences invol ved are

• Transformation of side chains.

• Formation of ring fission substrates and • Ring cleavage and transformation of the products

of ring fission into common compounds of intermediary metabolism.

In the aerobic process, molecular oxygen IS

involved in two ways.

Oxygen is incorporated in the aromatic ring by means of mono and di-oxygenases prior to ring fission2

.

2 Oxygen serves as the terminal electron acceptor of the reducing equivalents generated during the oxidation of the aromatic molecule.

In aerobic metaboli sm, ring fission occurs by a variety of bacteria, fungi and actinomycetes through mono or dioxygenases which incorporate either a single or two oxygen molecule to form reduced central intermediary , catechol. Catechol is converted to either pyruvate or acetaldehyde. The end products are further catabolised through tricarboxylic acid (TCA) cycle. The aerobic cleavage of aromatic ring is achieved through dioxygenases by three mechani sms :

I Ortho-fission 2 Meta fission 3 Gentisic acid path ways.

In ortho-fission pathway , ortho-dihydric phenols are cleaved in between two neighboring hydroxylated carbon atoms, resulting in the formation of dicarboxylic acids, which enter the intermediary metabolic pathways through their CO-A derivatives.

1050 INDIAN J EXP BIOL, SEPTEMBER 2003

Meta fi ss ion is the cleavage of the ring between the hydroxylated and non hydroxylated carbon atoms. In case of gentisic acid it is cleaved at the carbon-carbon bond between the carboxyl groups and the neighboring hydroxyl group to g ive maleyl pyruvic acid followed by a glutathione dependent isomerization to form fumaric and pyruvic acids.

Anaerobic degradation of aromatic compounds (Ring cleavage without oxygen)

Anoxic conditions are generally created when oxygen consumption exceeds supply e.g. in soils with impeded drainage, stagnant water, municipal land fills, sewage treatment digesters, industrial plants that produce methane from organic waste, the alimentary tract of all animals, and finally sediments of the ocean and other natural bodies of water. Under these conditions, biochemical mechanisms, which do not involve molecular oxygen, take over, during the breakdown of aromatic compounds. As a consequence, some types of aromatic compounds make it difficult to be degraded anaerobically, in particular, the aromatic compounds without functional groups (benzene, toluene, naphthalene, etc.) . However higher chlorinated aromatic compounds (e.g. hexachlorobenzene) are degraded better under anaerobic conditions. Different types of compounds can act as alternative terminal electron acceptors in the absence of O2, such as sulphate, nitrate, sulphur, oxidized metal ions (e.g. Fe3

+ and Mn4+) 8, protons and

bicarbonate ions. Proton and bicarbonate reduction are primary electron acceptors employed in methanogenic environment. Phototrophic bacteria can also use aromatic compounds for carbon and electron requirement in the absence of O 2, if light is available. In thi s case, the degradation product is a biomass rather than carbon dioxide9

.

Though the energy gain from aerobic degradation is hi gh, there are certain inherent characters of phenol s which auto oxidi se into macromolecules in the presence of molecular oxygen or light. Eismann

et al.10 observed that such molecules hamper the progress of degradation and only monomers are attached by bacteria. The auto-oxidation process is circumvented in anaerobic conditions where the existing reducing conditions prevent the phenol s to condense into humus like macromolecules. Another significant factor is that in many ecological niches, aerobic and anaerobic states often exist together since the solubility and penetration of oxygen is low. These coupled with the evidence for the degradation of hydrocarbons and other phenolic compounds found in anaerobic environments, led to intensive investigation in this area. The fundamental differences in aerobic and anaerobic degradation of phenolic compounds3

are outlined in Table 3.

Anaerobic environments Anaerobic conditions commonly exist in soil s and

sediments. Anaerobic degradation processes are important factors cons idered in the design of sewage digesters and municipa l landfills.

Soils and sediments Well-aerated soil s are considered to provide the

best condition for plant growth and agricultural productivity. In waterlogged or compacted soil s, anaerobic reactions become correspondingly more important. In contrast, sediment environments are influenced by the conditions of the water column above~ If oxygen is not depleted from the water, most sediment are aerobic for first few centimetres. Under oxygen poor conditions, sediments can readi ly become anoxic. If continued, biolog ical oxidation of organic compounds is to occur, the potential ro le of alternative electron acceptors such as nitrate, sulphate and carbonate must be considered.

The importance of anaerobic processes in the carbon cycle and in the degradation of organi c compounds in the environment can be illustrated by several investigations. In a New England salt marsh, the surface mediated respiration of organic matter was

Tabl e 3 - Compari son of ae robic and anaerobic aromatic mctaboli sm

Step

Channeling

Central intermedi ates Properties of centra l intcrmcdiates Attack a t the ring Ring c lcavage Path way to central metabo lic

Aerobi c

Oxygen

Catecho l, proto catechuate genti sate Easy to ox idi ze (c leave) O2

Oxygenolys is o f aromatics 3-oxoadipaterrCA cyc le

Anaerobic

+H20 . +2(H), -2(H), +(H20), +C0 2,

+C0 4 , +ATP Benzoy l CoA, resorcinal phl orog luc ino l Easy to reduce (hydrate) 2 or 4( H) + (H20 ) Hydro lys is of 3-oxocompouncl . B-ox idati on

f

JOTHIMANI el al.: ANAEROBIC BIODEGRATION OF AROMATIC COMPOUNDS 1051

12 times higher than that mediated by oxygen. In terms of methanogenic contribution , For a fresh water lake in Michigan as much as one third of the total primary producti vity could be recovered as methane over a summer season3

. Thus the contribution of anaerobic organisms to the recycling of organic matter is not minor and warrants more attention .

Digesters Anaerobic digesters are used in munic ipal sewage

treatment plants. Thi s condition is a highly reducing environment, which permits the mineralization of organic compounds to CO2 and CH4. Anaerobic digesters require relatively restrictive environmental conditions. Nevertheless they are known to contain a large and di verse microbial population. Within the last few years, new strains of methane bacteria have been isolated from laboratory digesters. Several of the new isolates are unusual in not being able to medi ate CO2 reduction with hydrogen. A wide range of substrates enter in digester, this necessitates a functionally diverse population of microorgani sms that is capable of reducing inorganic nitrogen and sulphur compounds. The population must also be capable of degrading lipids, cellulose, variety of protei ns, carbohydrates and other complex polymers. Thi s is in contrast to the microorgani sms in the rumen whose substrates consist of a more limited range of polymers such as cellulose, starch and pectin6

.7

.

Two functionally defined populations are responsible for the complete conversion of complex organic pol ymers to CO2 and CH4. The two populations are mutually dependant since continued activity by the first group, acid forming bacteria requires the removal of the organic ac id products by the second group, the methanogens. In digesters, more than the 70% of the methane originates from acetate, with the remainder coming from other sources. Third group are the hydrogen producing acetogenic bacteria. Their fun ction is to convert organic alcohol and acetate with the reduction of protons to hydrogen.

It should be emphas ized that acetate or hydrogen produciJ;1g species may playa key role in supplying the hydrogen for the reduction of CO2 to CH4. Thi s is illustrated by the studies of Bryant and hi s colleagues on Methanobacillus omelianskii. This culture was shown to consist of a syntrophic assoc iation of two species of bacteria. One bacterium metaboli zes ethanol to acetate and hydrogen.The second organi sm is a true methanogen that uses the hydrogen to reduce

CO2 to CH4. It is clearly evident that multiple species interactions are essential for the decomposition of complex organic materials and for nutrient turnover in these anaerobic systems. hence the class ic model of a single substrate-single organism is not applicable.

Landfills Sanitary landfills are a means of disposing of the

solid waste produced by municipalities. When wet garbage, pl astics, grasses, and woody materials are considered, it can be seen that upto 88% of the waste is organic and theoretically fermentable . The anaerobic digestion occurring in landfill s produces methane in an uncontrolled environment when compared to the more carefully controlled waste treatment reactors and therefore produces less than optimal amount. Gases produced at a given si te may continue for decades, and methane production rate have been reported to range from 0.06 to 0.23 SCF/IblY ear. The bas ic characteristics of microbial degradation would be expected to be similar to those described for digesters.

Gut and rumen The rumen is not unlike the other environments. It

operates at an oxidation reduction potential of - 350 mY , at which the oxygen concentration can be calculated to be less than 10-22 M. Substrates are fermented to organic acids, and methane is generated from the products of fermentation. The greatest source of methane is from CO2 reduction and formate metabolism. The acetate fermenting methanogens playa much smaller role in the rumen than they do in other systems.

Anaerobic degradation energetic The lack of O2 in the anaerobic environment leads

to the use of certain other potenti al ox idants. An alternative oxidant must be a compound, which can be reducible by a biological system, such that energy can be harnessed by the organisms and neither the ox idati on nor its reduced product is tox ic at the concentrati ons required J J. In anaerobic environment,

CO2 or CO ; , NO ; of N02· and SO ~ can be used as

an alternate electron acceptor depending on the respiratory capabilities of the surviving bacteri a, the corresponding environments being methanogeni c, nitrate reducing and sulphate reducing processes respecti vell . It is known that degradation of benzenoid occurs in all these environments9

. The free energy for anae robi c conversion of benzoate, a major intermed iate , has been worked out by Field et al.4 .

1052 INDIAN J EXP BIOl, SEPTEMBER 2003

Reaction

Benzoate + 7.5 O2 ~

Benzoate +6 NO ;- ~

Benzoate +3.75 NO ;- ~

Benzoate +30 Fe+ 3 ~

Product

7 CO2

7 CO2 + 3 N02

7 CO2 + 3.75 NH4

7 CO2 + 30 Fe2+

Gibb 's free energy(KJ)

-3 174.6 - 2977.3

- 1864.3

- 3043.3

Benzoate +3.75 SO ; - ~ 7 CO2+ 3.75 HS' -185.4

Benzoate +25 So ~ 7 CO2 + 15 HS' -35.8 Benzoate ~ 3.25C02+ 3.75 CH4 - 124.3

The maximum energy gain occurs in aerobic conditions, followed by ferric and nitrate as respiratory oxidants. In sulphur reducing and methanogenic environment, the energy gain is very low. The effect of different electron acceptors intoluene degradation was studied by Eismann et a/.IO. They found that the maximum degradation occurred in nitrate reducing conditions. ]n contrast, methanogenic and sulphate reducing environments registered almost no degradation at all. Thauera aromatica, Azoarcus evansii and Rhodopseudomona palustris showed maximum benzoate degradationt2. Recently sulphonates have also been found to act as an electron acceptors 13, but their implications on anaerobic metabolism of aromatic compounds are not known. Although a few reports on Pseudomonas sp. are documented, the non-availability of the physiological nature of growth prevents a valid conclusion. Jorgensen el a/. 14 provided evidence for nitrate conversion and correlated the data with toluene degradation.

In spite of the differences in energy gain, the end product of the process seems to be either benzoate or hydroxyl benzoic acid as postulated by Evans 15 but these exist as Co-enzyme A thiesters rather than as free acids l6

, which are then subjected to a reductive path of degradation.

Anaerobic lifestyles - Electron acceptors Metabolism in the absence of oxygen is dominated

by the nature of the available electron acceptor or hy­drogen sink. Successi vely, nitrate, ferric iron, sul­phate, and carbon dioxide serve as the preferred elec­tron acceptor for the denitrifying, iron reducing, sul­phate reducing, and methanogenic bacteria. Environ­ments of the terminal electron accepting processes (TEAPs):

Electron Acceptor TEAP Environment

O2 Aerobic SedimenllGW

NO ] Oenitrifying Nitrate-rich GW

Fe(lll ) Iron reducing GW

Sulphate reducing Marine systems

Methanogeni c Organic rich systems

It also appears that oxidized groups on humic substances can serve as electron acceptors. Humic acids may act as redox shuttles between immobile ferric iron and bacteria in suspension . In addition , acid-forming fermentative bacteria and H2 -producing acetogenic bacteria are a part of many anaerobic communities:

In anaerobic environments oxygen is not available to promote activation of recalcitrant subs trates, such as aromatics.02 has two functions in metaboli sm: As electron acceptor and as a reactant in oxygenase reactions.Until recently it was thought that only substituted benzenes were degradable without oxygen. The substituents such as carboxy l, methyl , or hydroxyl decrease resonance energy by withdrawing electrons and destabilizing the ring and making ring cleavage easier. In the last few years toluene, xylenes, benzenes, and PAHs have been shown to be degradable.

Principle steps in ring cleavage without oxygen Three types of reactions :

1. Activation reactions (examples of activation reactions)

p-Cresol-p-hydroxybenzyla\Cohol-methyl hydroxylation of p-cresol

Toluene-benzy la\Cohol-methyl hydroxylation of toluene

Benzene-phenol-hydroxylation of benzene ring Toluene-hydrocinnamoyl-CoA-methyl grolJlp

oxidative addition

2. Channeling reactions to central intermediates

3. Ring cleavage reactions-Ring cleavage and p-oxidation reactions to form common intermediates for energy production and cell synthesis - Following production of central intermediates, the ring is cleaved to produce aliphatic acids, leading to common metabolic end products (C02, C~, and biomass). These generally involve three reactions:

1 Reduction of the aromatic ring to an alicyclic ring 2 Formation of cyclohexanone 3 Hydrolytic cleavage

Anaerobic metabolism-General pathways Cfable 4) The pathways of degradation of aromatic acids,

phenols and hydrocarbons has been made through the study of,

y

JOTHIMANI el al.: ANAEROBIC BIODEGRATION OF AROMATIC COMPOUNDS 1053

a. The photosynthetic anaerobic metabolism of benzoate.

by an identified consortium of bacteria co­operating to form a food chain.

b. The anaerobic metabolism of aromatic acids and phenols by nitrate respiration .

Methanogenic degradation of naturally occurring aromatic compounds

c. Anaerobic dissimilation of aromatic compounds through sulphate respiration .

All known methanogenic species are found fastidious and can use only a limited number of s imple carbon substrates such as formate, methanol , methyl amine, acetate and carbon dioxide. Hydrogen is used as the electron donor for CO2 reduction . This energy yielding reaction results in the formation of

d. Anaerobic fermentation of many polyphenolic substances and

e. The methanogenic fermentation of almost all naturally occurring soluble aromatic compounds

Table 4 - Major groups in aromatic metaboli sm9.17

Energy yielding process

I. Photosynthetic

2. Phosphory lation

3. Nitrate respiration

NO ; + 2W + 4H2 ---)

NH ; + 3HP

~Go ' = -600K]

4. Sulphate reduction

SO ~2 + 2H+ + 4H2 ---)

H2S + 4H20 ~Go' = -IS2Kl

S. Fermentation

6.Methanogenic fermentation HCO)' + H+ + 4H2 ---)

CH2 + 3H20 ~Go' = - 13S K]

Organisms

Rhodopseudomonas paluslris,

Rhodopseudomonas gelalinosa

Pseudomonas PN-I (Alcaligenes xylosoxidans) Moraxella sp. (Paracoccus denilrijicans) Bacillus sp.

Pseudomonas Desulfovibrio sp. Desulfococcus Desulfonema Desulfosarcina

Coprococcus sp. 51 replococcus Pelobacle,. acidigallici Eubacterium oxidorens

Microbial consortia: Fermentati ve bacteria Acetogenic and Methanogenic bacteria

Substrates

Benzoate

m, p - Hydroxybenzoate phloroglucinol

Benzoate H ydrox ybenzoates Vanillate 0, m, p - Phthalate 2-Amino benzoate Phenol, 0, m, p - Cresol

Benzoate Hydroxybe nzoates Phenol acetate, hippurate Phenol, indo l

Phloroglucinol Resorcinol/acids Gallate, pyrogallol Polyphenols, quercetin

Lignin Benzoate Tyrosine Cinnamate Phenyl propionate Phenyl acetate. Phenol, catechol Hydroqu inone Ferulate Vanilate Syringate Benzoate Phenylalanine Tyrosine Tryptophan , indole Conifery l alcohol Benzene, toluene Chlorobenzene Ch lorophenols Chlorobenzoates Chlorophenoxyacetates Nitrophenols Chloroguaiacol s

1054 INDIAN J EXP BIOL, SEPTEMBER 2003

methane. Because of these limited capabilities, the metabolism of more complex molecules to methane depends on the activity of non-methanogenic counter part to convert the complex molecules to substrates for methanogens. Methanogenic consOItia metabolizing lignin and related complex molecules are g iven below (Table 5). Examples of metabolic pathways19

Anaerobic degradation of benzoic acid Anaerobic degradation of benzoate proceeds by

two pathways: via pimilic acid and adipic acid both

involve reduction (saturation) of the aromatic ring and incorporation of an oxygen from water leading to an enol prior to ring cleavage. By reduction of the aromatic ring the resonant electron di stribution is destabili zed. The phototrophic purple nonsulfur bacterium Rhodopseudomonas attacks benzoic acid by th~ pimilic acid pathway (Fig. I) , using coenzyme A as a cofactor: Denitrifiers, a Moraxella sp. and Pseudomonas PN I, degrades by reducti on of the ring, followed by decarboxylationas in the following path way (Fig. 2) . Sulphate reducers are also ab le to

Table 5 - Methanogenic consorti a metaboli zing li gnin

Compound

Li gno cellulose

Cowdung

Coi r dust li gnin

Intermedi ates

Ci nnamate, benzoate, ca feate, vanillate

Benzoic ac id , hydroxy benzoic ac id, protocatechui c acid. cinnamic acid , acetate, butyra te, va lerate

Cinnam ic acid , ferulic acid, vanilli c acid, hydroxy benzoic ac id , acetate, propionate, butyrate, valerate, caproate

Benzoic Acid

Ben7.0yl -CoA

"-~--- ~--~) y Redllc/ive phase

2-hydroxycyclohexane

Carboxyl-CoA

COA lIIedia/ed fJ- oxida/ion phase

Organi sms in volved

Consortium

Consortium

Consortium

Pirrclyl-diCoA

COSCoA

~6=cM 3-Hydroxyl-pinl'!l yl -diCoA

Fig. 1- Pimilic ac id pathway using coenzy me as a cofacto r

Reference

18

17

17

JOTHIMANI el al.: ANAEROBIC BIODEGRATION OF AROMATIC COMPOUNDS 1055

degrade benzoates, but not through cyclohexanol or cyclohexanone (Fig. 3). No pathways for aromatic metabolism by sulphate reducers are published so far. Anaerobic degradation of phenol is shown in Fig. 4 . Note that complete degradation of aromatics under methanogenic conditions require interaction of a consortium. Sulphate reducers may take the place of methanogens as accepting hydrogen in consortia degrading aromatics (Fig. 5).

2 Formation of a cyclohexanone or ethylcyclohexanone 3 Reductive cleavage of the ring to aliphatic acids

All of these pathways involve ] Reduction of the ring, often involving coenzyme A

4 Degradation of the cleavage products to compounds suitable for methanogenic use.

Anaerobic degradation of lignin Lignocelluloses makes up to about 95% of the

earth's renewable, land-produced biomass ; about one quarter of this is made up of aromatic polymer lignin . Anaerobic degradation occurred when the solubilized lignin fractions were incubated with' an inoculum from

6::H

. 3H, ;::H, ~ .H,O 6COOH

OH 6COOH

0 --~.~~ ~~ ~ ~

# -H2 ~-C02 00 Benzoic 2-Hydrox ycyclohexane

\. Acid ) "- carboxylic ac id y ~----------

Redllctive phase Coenzyme-A mediated

CHO -H OCH2~~"''''0I-H __ ?<r: H, OH

O OOH ~ . H,o ~. _________ COO H

Adi pic Acid --------. O OO H ---1~. Melabo lic

Pool ---~. Acelale. H2. cO2

Fig. 2 - Benzoic acid degradation through adipic acid path way

1.3-Cyclohexanedione

Fig. 3 - Cyc lo hexanol degrada ti on

1056 INDI AN J EXP BIOL, SEPTEMB ER 2003

an anaerobic, mesophilic sludge digester; after 30 days the fraction with the highest molecular weight was much reduced. The reduction was attributed to cleavage of the inter monomer bonds duri ng anaerobic attack, which produced lignin monomers readiliy degraded to methane and C0 2

20. Other compounds, significant in degradation

of lignin that occurs naturally and in pulp and paper making, can be anaerobically degraded: Catechol, vanillin , syringic acid, syringaldehyde, ferulic acid,

cinnamic acid, p-hydroxybenzoic ac id, protocatechuic acid, and pyrogallol, a product of lignin degradation (Fig. 6). All requ ire a reduction of the aromatic ri ng, fo rmation of cyclohexanone, then a cleavage leading to a dicarboxy li c ac id, as fo r catechol (Fig. 7) .

Anaerobic degradation of phthalates Phthalate acid esters are important as pl asticizers

(Fig. 8) . They, leach out of pl astics and are a major

OH OH 0

6 6 6 +3 H2 · H2 +HP • •

or through benzoate

6 OH

¢ H OH

+c02 +Hz • Q -Hp

• COO H COOH

Fig. 4 - Phenol degradation

UOOH Pimelate

+H 2~

COOH COOH CHI /UCHJ

6 6 0 60 COOH ...... ...... ---. I # +3Hz +HzO

Benzoic ~ Heptanoa tc Acid -C02

O OH 00 OCO~~, I # ...... +HP. .

Phenol Cyclohexanone

~ \t

II-Caproate

COOH OCOOH

Adip ic Acid

•

•

COOH UH' ___ .~ Metabo li c Pool

II -caproate

0 COO H

Metabolic ---1.~ Pool

Va lerate But yra te

~ Propionatc ~ -7 Acetate -7

Formatc Hydrogen

Fig. 5 - Benzoic ac id degradation by sulphate reducers

JOTHIMANI el al.: ANAEROBIC BIODEGRATION OF AROM ATIC COM POUNDS 1057

contaminant of municipal sludges. Bis(2-ethylhexy l) phthalate accounts for abo ut 23% of phthalate production and it is a suspected carcinogen. Phthalate esters are degraded slowl y under anaerobic conditions with less degradation for lo nger side-chains: 100% loss in 7-42 days for methyl, e thy l, butyl, and butylbenzylphthalates , but in sig nificant loss of octy l and ethylhexylphthalates. The proposed pathway for the degradable phthalate esters is by cleavage o f a side chains sequenti ally , fo rming a -phthalate, the ring cleavage by an unknown pathway (Fig . 9) . Phthalic acids degraded by decarboxyl ati o n through benzoy l-

CoA and pimelate as in the degradation of phthalates by a denitri fy ing bacterium (Fig. 10). Anaerobic pathways leave hexylethy lphthalate untransformed in digested municipal sludge.

Pyrogallol Ph lorog lucinol

Anaerobic degradation of cresols Para-cresol was degraded rapidly in accl imated

sediment systems under denitrify ing, sulphate reducing, and methnogeni c conditions, but took 3-4 weeks in unacclimated sediments . It was suggested that the same initi a l pathway was used fo r p-creso l metabo li sm: via oxidation of methyl substituent to

Fig. 6 - Pyroga ll ol degradation

Catechol Cyclohexanone

¥

Succinate CJCOO~OOH¥

.- .- Propionate .-- Adipic acid Acetate

Fig. 7 - Catechol degradation

OtO-R

1 R =

~ C - O - R II o

CH1• methyl CH2CH3• eth yl (CH2)3CHJ' butyl (CH2)5CH3' hexyl -0. benzyl

Fig. 8 - Structure of phthalates

~ ~ ~

0 1 C - O -~Ol C - O -~Ol C- OH ---. C: .

~ C - O - R ~ C - OH ~ C - OH CO2 II II II o 0 0

Phtha late Ester (PAE) Mono phtha late Ester (PAE) Phthalic ac id (PA)

Fig . 9 - Phthalate es ter degradation

1058 INDI AN J EXP BIOL, SEPTEMBER 2003

p-hydroxybenzaldehyde and p-hydroxybenzoate, both of these in termedi ates degraded rapidl y in both acc limated and unacclimated sediments. Benzoate appeared as an inte rmedi ate in methanogenic cultures, but not in denitrifying or sulphate-reducing (Fig . 11 ).

Anaerobic degradation of toluene and xylenes The BTEX chemicals, benzene, to luene,

ethyl benzene and a-, m-, and p -xy lene are major contaminants of so ils and gro undwater, o ri ginating mainl y fro m leaking underground gaso line tanks. (Fig. 12). Some studies have found evidence fo r degradati on of a ll of these compounds, but only to luene and m- and a-xylene have been studied with pure cultures. Both denitrifying (strain T I ) and an iron reducing strain (strain GS-IS ) have been isolated that can grow o n to luene and a-xylene and can

transform m-xy lene 7 , 8. The pro posed path way is through ox idatio n of the meth y l substituent to benzoy l coA th ro ugh the in vo lvement of acetyl coenzy me A, with concomitant formati on of benzy lfumaric and benzy lsuccinic ac id as dead end metabo lites in vo lving sLi cc inyl coenzy me A as shown in Fig. 13. In mi xed methanogenic cultures using 14C-labe led to luene, labe led benzene, pheno l, and a-and p-creso l have been o bserved as we ll as methy lcyclohexane and benzoic acid . Labeled benezene led to recovery of cyclohexene, cyclohexanone and pheno l.

P AH degradation under anaerobic conditions Degradation of naphtho l, naphthalene, and

acenaphthalene (Fig. 14) under denitri fication conditions, but not sulphate-reducing conditi ons, has been observed in fresh water-so il slurries using

6 e OOH ~ 6 00

"

ATP ADP+P ~o, , e OSCoA . caSCoA caSCoA COSCoA

CuASH co COOH

I ~ I ~ 2 2. I ~ ----+ ~ ----+ OH

COOH

ATP AOP.Q COOH

¢ COOH B Oxidation

Fig. 10 - Phthalic acids degradati on

COOH o ~ ~ 0" __ Q --c5 M,,""""'''''C • 6 OH OH OH OH

/J' hydroxybenzaJdehyde /J - hydroxybenzoate

I I

DellYlrtfYill~ SlI lJidogellic

Fig. I I - Cresol degradati on

Ri ng reductiun Ring f iss ion

JOTHIMANI el al.: ANAEROBIC BIODEGRATION OF AROMATIC COMPOUNDS 1059

radiolabeled PAHs and recovery of CO2. Naphthalene, phenanthrene, and biphenyl were degraded under nitrate- and sulphate-reducing conditions III

enrichments from manne sediments (Fig. 15).

Carboxy-derivatives have been reported to be the first intermediate in the degradation of naphthalene and phenanthrene in a sulphate-reducing enrichment culture, but the activation mechanism is unknown .

0 Benzene

6 0"' Toluene Ethylbenzene 0- Xylelle

III - Xylell e p. Xylelle

Fig. 12 - Structure of toluene and xylene

Propinyl CoA

COOH I

CH2 C=CH I COOH

Fig. 13 - Oxidation of the methyl substituent to benzoyl coA

OH

ex) 0::) Naphthalene Naphthol Acenaphthalene

Fig . 14 - Structures of naphthalene, naphtho l and acenaphthalene

00 --C:a~----'. 00= -+ CO,

2-Naphtholic Acid

Fig. 15 - Naphthalene , phenanthrene, and hiphenyl degradation

Dead End

1060 INDIAN J EX P BIOl. SEPTEMBER 2003

Chloroethene mineralization under anaerobic conditions

Vinyl chloride and cis-l ,2-dichloroethylene have been reported to be degradable to carbon dioxide under anaerobic conditions with ferri c iron , humic ac ids, sulphate as electron acceptor and with methanogenesis.

Bioremediation of aromatics using anaerobes (Table 6)

Many microorgani sms are capable to transform or degrade the nitroaromatic co mpounds. Such orga ni sms are candidates for use in bioremedi ati on. The use of microorgani sms to degrade aromatic pollution is an interesting low-cost approach for the treatment of industri al effluents, contaminated sediments in so il and ground water. The vast number of tudi es in thi s field particularly in the degradation of li gnin by whiterot fungi . resulted in complete understanding of metabolism, enzy mes and genes in vo lved l.2.

Typicall y, bioremed iation is practiced by providing a favo urab le environmental conditi ons in order to sti mul ate the degradation of pollutants by naturall y occurring mi croflora. Recentl y Oagher et 0/.

2 1 carri ed out the comparati ve study of five polycyc lic aromati c hydrocarbon degrading bacterial strains (Pseudomonas putida 34. P. Fluoresens 62, P. Aeruginosa 57, Sphillgomonas sp. strain 107), and the unidentified strain PL I isolated from contam inated soils. Several genes encoding enzy mes in volved in the upper

catabolic pathway of naphthalene were present in each strain . These genes are highly homologous with respective genes found in the pah, dox and nah operons and are arranged in a polycistronic operon. Likewise many attempts have been made to rec laim soil sites contaminated with phenolic and other aromatic compounds.The biodegradati ve capac iti es must be developed by carefull y selecting the correct combination of electron donors , electron acceptors and in the case of cometabolism, the choice of the pri mary substrate. Additi onall y, the bioremedi ati on usually entail s the supplimentat ion of limiting nutrients.

The selec tion and improvement of anaerob ic phenol-degrading consorti a in sewage sludge to produce a max imum degradati on rate - The process was studied in va rious cond itions using rad ioisotope­labelled phenol. I n the presence of a hydrogen:carbon-diox ide atmosphere turnover rates of phenol were significantl y reduced and benzoate was formed from phenol and CO2. The accumul ated benzoate was subseq uentl y degraded to methane and CO2, Hi gh levels of acetate in sewage sludge also led to a reduction in phenol-degradation rates and probably to an increased concentrat ion of benzoate.

Under the appropriate conditions, BTEX components of petroleum (benze ne, toluene, ethylbenzene and xylene) can be degraded in the absence of molecul ar oxygen with either Fe( III ). sulphate, or nitrate serving as the electron acceptor. BTEX degradati on under methanogeni c conditions

Table 6 - Bioremed iati on using anaerobes

Habi tal

Underground

Freshwater mud samples Fue l contaminated aqu i fers Aqu i fers. lake and sa lt marsh sediments ESlLIarine sed iment Cowdung sludge and municipal waste water Ircatmenl planl Sewage treatmenl plants Flooded soil s Municipal so lid wastes

Anaerobic soil column Aquifers Swine manure

Compou nd

2.4-dipheny lacetie acid. 2A.3-T .M CPA A lky l benzene BTEX

Carbamalcs

Halog inated phenols Benzene toluene

To luene Fluchlorin Chlorobenzenc deri vat i ve: Tri nitro to luene Phenols & toluene Phenol M onocyc li c aromat ic compounds

Organi sm

Insilu organi sm

Thauera se lenali s Bacterial consortium

Bacterial consortium

Bu l fidoge nic consorti um Bacterial consortium

Bacteri al consortium Flooded so il microfi ora Bacterial consortium Anaerobic spore forming soi I bacteri a Burkholderi a cepacia G4

Bacterial consortium Facul tative bacteria

Environment

Vari ableredox eondili ons

Nitrate reducing KNOJ . denitrify ing

Nitrate reduci ng

Su lphale reducing Su lphate reduci ng

Deni trify ing

A noxic A noxic

Anox ic Reducing & methanogenic

Reference

22

2J 24

25

26 27

14 28 2Y 30

31 32 33

JOTHIMANI et al.: ANAEROBIC BIODEGRATION OF AROMATIC COMPOUNDS 1061

has also been observed . However, for benzene, the BTEX contaminant of g reatest concern , anaerobic degradation is often di fficult to establi sh and mainta in in laboratory incubatio ns. A ltho ugh studi es to date have suggested that natura ll y occurring anaerob ic BTEX degradatio n has the potentia l to remove signi ficant quantities of BTEX fro m pet roleum-contaminated aqui fers, and mechani sms fo r stimulating anaerobic BTEX degradatio n In laboratory incubations have been developed, further study of the organisms in vo lved in thi s metabo li sm and the factors controlling the ir di stri buti on and acti vity are requi red before it will be poss ible to design rational strategies fo r acce lerating anaerob ic BTEX degradation in contaminated aqui fe rs34

. A lso enriched sedi ments fro m 4 marshes in the National Donana Park , Spain ,were cul tured in the presence of ferulic acid as the o nly source of carbon and energy to determine the metabolic inte rmed iates resulting from anaerobic degradation. The fo llowing compo unds were identif ied by HPLC analyses: caffe ic ac id , coumaric acid, p-hydroxy pheny lpropio nic acid, p-hydroxybenzoic ac id and catecho l. The presence of catechol resulting fro m the hydroxy lation of phenol suggests that thi s metabo lic response could be utili zed by the bacteri a as a mechani sm of detox ificatio n in a marshy environment.

2,4-Dichlorophenol (2,4- DC P) was anaerobically degraded in fres hwate r lake sediments. Fro m observed intermedi ates in incubated sediment samples and from enrichment cultures, the fo llow ing sequence of transformations was postul ated: 2,4-DC P IS dechlorinated to 4-chloropheno l (4-C P); 4 -CP IS dechlorinated to phenol; pheno l is carboxy lated to benzoate; and benzoate is degraded via acetate to methane and CO2; at least 5 di ffere nt organi sms are in volved sequenti a lly. The rate-li miting step was the transformation of 4-C P to pheno l. Sediment- free enrichment cul tures were obta ined which cata lysed on ly the dechlorinatio n of 2,4-DCP, the carboxy lati on of pheno l and the degradatio n of benzoate, respecti ve ly. Whereas the dechl orinati on of 2,4-DC P was not inh ibited by H2, the dechl orinati onof 4-C P and the transformatio n of pheno l and benzoate were. Low concentration of 4 -CP inhi bited pheno l and benzoate degradati on. T ransformati on rates and maximum concentrati on a ll ow ing degradati on were determined in both fres hly collected sediments and in adapted samples: at 3 1 deg ree Celc ius which was the optimum temperature fo r the dechlorinati on, the average adaptation ti me fo r 2 ,4-DCP, 4-CP, phenol

and benzoate transformations were 7, 37 , 11 and 2 days, respectively T he maximum observed transfor­mation rates fo r these compounds da ily in acclimated

sediments were 300, 78 , 2 130 and 2080 j.lmole/litre, . I 35 respectI ve y .

Degradation of insecticides in flooded soil ecosystem More rapid degradation of Lindane (r-BHC)

occurred in unste rili zed fl ooded soil than in sterili zed fl ooded so il indicating bio logical partic ipation. Clostridium sp., iso lated from lindane-amended fl ooded so il , metabo lized r- and a-BHC, anaerob i­ca ll y, but not Band b-i somers which were the rmodynami ca ll y more stable. The presence of nitrate o r oxygen in the incubati on mi xture, inhi bited bacteri al degradation of Iindane36

. During bacte ri al degradati on of ring labe led-r and a -BHC, a rad io acti ve compound with strong e lectron affinity was fo rmed. Thi s metabo lite di sappeared from the incubati on mi xture rapidly .

T here are two main path ways in the degradation of lindane (C6H6Ci6) Degradation via dehydrochlori­natio n leading to the fo rmatio n of C6H5C Is as in insects and mo ist so il s o r w ith two unicellular algae, Chlorella vulgaris, Beijerinckia and Chlamydomonas reinhardtii and Degradation via reducti ve dechlo ri­nat io n in which one chl orine ato m of lindane is substi tuted by one hydrogen ato m to fro m C6H7Cis as speculated in the anaerobic decomposition of lindane by Clostridium Sp.37 .

In the case of DDT, the add itio n of organic materi a ls to flooded so il or to an anaerobic so il enhanced DDT conversion to T OE. In an in vitro anaerobic system. rumen microorganisms converted O,P ' - P, P ' - DDT to 0, P ' - and P, P' T OE -respecti vely and to an unidentified po lar metabolite. Methoxychlor was mo re biodegradable than DDT under anaerob ic condition . Because of the lower affinity fo r fa t, methoxychl or looks promi sing as a substitute insecti c ide for DDT in fl ooded soil and anaerobic environments. Pesticide

Indigenous mixed popul ations of anaerobic microorganisms fro m and irrigation ta il water dra in and submerged ag ri cultura l chemical was te pit readily b iodegraded the major isomer of endosu lfan (endosulfan I). Endosuifan ] was biodegraded to endosul fa n dio l, a low tox ic ity degradati on product, in the presence of organic carbon sources under anaerobic, methanogenic conditio ns. While there was extens ive degradation (>85 %) over the 30 days , there was no sig ni ficant enh ancement of degradation from

1062 INDI AN J EXP BIOL, SEPTEMB ER 2003

enriched inocula. Thi s study demonstrates that endosulfan J has the potential to be biodegraded in sediments, in the absence of enriched microorgani sms. This is of particular importance -ince such sediments are prevalent in cotton-growing areas and are typically contaminated with endosul fa n residues . The importance of minimizing non­bio logical losses has also been highli ghted as a critical issue in determining anaerobic biodegradation potential. Seals for such incubation vesse ls must be both oxygen and pollutant impermeable. Teflon-lined butyl rubber prov ides such a seal because of its resistance to the absorpti on of volatiles and in preventing volatili zation . Moreover, including a 100 mM phosphate buffer in the anaerobic medi a has reduced non-bio logical losses from chemi cal hydrolysis, allowing biodegradation to be assessed38

.

Growth of sulphate-reducing bacteria on crude oil In addition to indi vidual hydrocarbons, crude oil as

a natural, complex mi xture of hydrocarbons was also tested as growth substrate for sulphate-reducing bacteria l 9

. A mesophili c enrichment culture from an o il tank was shown to utili ze alkyl benzenes from crude oil added as the onl y source of organic compounds to defined anoxic mineral medium. More than 95% of the enriched population was members of the suggested family of the Desuljobacteriaceae. Members of thi s family of sulphate-reducing bacteri a are distincti ve from Desuljovibrio and Desulfomicrobium species. Thi s observation is in agreement with the finding that many sulphate­reducing bacteria that degrade aromatic compounds are members of the Desulfobacteriaceae.

Growth of denitrifying bacteria on crude oil Research on anaerobic degradation of o il

hydrocarbons especially by denitrifying bacteri a has been stimulated by the fact that dispersal of oil and fuel in the environment may lead to the contamination of deep aquifers. Since groundwater aquifers are frequently anox ic, knowledge of bacterial capacities for degradation of oil hydrocarbons in the absence of molecular oxygen is important to predict the fa te of hydrocarbons and the effectiveness of bioremediation efforts under such conditions. Addition of nitrate to o il-contaminated sites, e.g. groundwater aquifers, has been regarded as a potenti al means of enhancing bi oremedi ation efforts on site. Furthermore, the study of anaerobic hydrocarbon oxidation by nitrate reducers may help to understand future side effects of

the suggested contro l of sul fide production during o il production by the addition of nitrate. Strains of denitrifying bacteri a which had been orig inally enriched and isolated on to luene, eth ylbenzene. propyl benzene, and m-xylene as defined substrates were shown togrow also on crude o il by utili zati on of alkylbenzenes. Toluene, ethylbenzene and m-xy lene disappeared completely during the first growth phase within 10 days, while o-xy lene, o-ethylto luene and 1,2,4-trimethylbenzene were partially consumed. During the second growth phase, parti al consumpti on of n-alkanes in the range of C5 -C I2, in particul ar of hexane, was observed . It could be calculated that 3.1 % (vol/vol) of the o il was anaerobica ll y ox idized by denitrification. alkylbenzene-degrading denitrifi ers related to Azoarcus species allowed the in situ detection of such bacteri a in subsurface soil ex posed to fuel hydrocarbons l9

.

Impact of environmental factors on anaerobic degradation

An investigation in vol ving the supplement of different concentrati ons of a substrate and mjcroorgani sms was conducted under anaerobic conditions to assess the concentrati on effects of the substrate and microorganisms on the in situ bioremedi ation of 1,1, I-trichloroeth ane TCA)4o. Experimental results demonstrated that the addition of higher concentrations of the substrate and microorganisms would enhance the biotransformation of TCA. The biotransformation rate of TCA increased progress ively with the increase of the concentrations of the substrate and microorgani sms. It is concluded that the bioremediation of groundwater contaminated with heav ily chlorinated hydrocarbons is feas ible in aqui fe r systems with appropri ate concentrations of substrate and microorganisms39

. Degradati on of fo ur heterocyclic compounds was examined under nitrate­reducing, sulphate-reducing and methanogenic conditions. So il samples from a creosote-polluted site in Denmark were used as inoculum. Indole and quinoline were degraded under all redox conditions with the highest degradation rates obtained under sulphate-reducing conditions. Benzothiophene and benzofuran were not degraded during the observation period of 100 days under any of the redox conditions. Indole and quinoline degrading cultu res could be repeatedl y transferred under all redox conditions, except fo r degradation of quinoline under sulphate­reducing conditions which was inhibited by sulphide at concentrations above 0 .8 mM. Degradati on of

JOTHIMANI et al.: ANAEROBIC BIODEGRATION OF AROMATIC COMPOUNDS 1063

quinoline under methanogenic conditions was also inhibited by 3.2 mM sulphide used as a reducing agent, but sulphide had no inhibitory effect on the degradation of indole in methanogenic and sulphate­reducing soil slurries4o.

The effect of cobalt on the anaerobic degradation of methanol by a mixed culture, was evaluated in a sludge with low background level s of cobalt in an upflow anaerobic sludge blanket reactor. Specific inhibitors in batch assays were used to study the effect of cobalt on the growth rate and activity of different microorganisms involved in the anaerobic degradation of methanol. Only methylotrophic methanogens and acetogens were stimulated by cobalt additions, while the other trophic groups utilizing downstream intermediates, Hr C0 2 or acetate, were largely unaffected. The optimal concentration of cobalt for the growth and activity of methanol-utilizing methanogens and acetogens was 0.05 mg/litre. The higher requirements of cobalt were assumed to be due to the production of unique corrinoid-containing enzymes (or coenzymes) by direct utilizers of methanol. Methylotroph .methanogens presented a 6-fold-higher affinity for methanol than acetogens. Acetogens grew slightly faster than methanogens under optimal cobalt conditions and indicated that acetogens can outcompete methanogens only when reactor methanol and cobalt concentration are high, provided enough inorganic carbon is available41 .

The degradation of benzaldehyde in methanogenic granular sludge wasinvestigated in batch and in upflow anaerobic sludge blanket (UASB) reactors . The effect due to the presence of co-substrates, such as H2, sodium butyrate and sucrose, was studied usi ng formaldehyde as a reference compound. The additional substrates enhanced the activity of benzaldehyde- and formaldehyde-degrading

. . 4? mlcroorgamsms -.

Anaerobic degradation of 2,4-dichlorophenol (2,4-DCP) at 5° -noc was investigated. Anaerobic sediment slurries prepared from local freshwater pond sediments were partitioned into anaerobic tubes or serum vials, which then were incubated separately at the various temperature. Reductive 2,4-DCP dechlorination occurred only in the temperature range between 5° and 50°C , although methane was formed up to 60 0 C. In sediment samples from 2 sites and at all tested temperature from 5° to 50° C , 2,4-DCP was transformed to 4-chlorophenol (4-CP). The 4-CP intermediate was subsequently degraded after an extended lag period in the temperature range from

15° -40°C. Adaptation periods for 2,4-DCP transformations decreased between 5° and 25° C, were essentially constant between 25° and 35°C, and increased in the tubes incubated at temperature between 35° and 40°C. The degradation rates increased exponentially between 15° and 30°C, had a second peak at 35°C , and decreased to c. 5% of the peak activity by 40°. In tubes from one sediment sample, incubated at temp. > 40°, an increase in the degradation rate was observed following the minimum at 40°C. This suggests that at least 2 different organisms were involved in the transformation of 2,4-DCP to 4-CP. Storage of the original sediment slurries for 2 months at 12°C resulted in increased adaptation times, but did not affect the degradation rates43

.

Molecular mechanisms of genetic adaptation to xenobiotic compounds

Different aromatic degradation pathways genes includes meta cleavage pathway genes, viz Extradiol dioxygenases; ortho cleavage pathway genes viz genes of the modified OItho cleavage pathways and lndradiol dioxygenases; genes for peripheral enzymes, viz multicomponent aromatic ring dioxygenases, monooxygenases and dehalogenases; and regulatory genes. Various mechanisms of genetic adaptation are gene transfer, point mutations; recombination and transposltlOn includes DNA reaJTangements , gene duplication, transposition and insertional activation.

For the understanding of adaptational processes in nature, in situ genetic interactions among microorganisms and the influence of environmental parameters on the selection and dissimination of specific catabolic genes should be studied. Many studies address the possibilities and frequencies of gene transfer under various environmental conditions. In meso cosm experiments, catabolic plasmid pBRC60 encoding 3-chlorobenzoate degradation of AlcaLigenes sp . Strain BR 60 was transferred to and expressed in indigenous recipient bacteria. The addition of low concentration of 3-chlorobenzoate, 4-chloroaniline, or 3-chlorobiphenyl to microcosm inoculated with Alcaligenes sp. strain BR 60 significantly affected the population size of microorgani sm that finally obtained Tn 5271 44 . To assess the importance of gene flow in nature, its rate has to be measured under environmental conditions. For example the transfer frequencies of conjugation of an epilithic plasmid encoding mercury resistance

1064 INDI AN J EX P BIOL, SEPTEMB ER 2003

(PQMl) were about 100 fold lower in microcosm studies that fitter mating experiments45 . The d istributi on of specific genes in natural environment can be determined by hybridi zation of DNA from indigenous so il microorganisms with spec ifi c gene probes. The technique allo wed a hi ghly specific detecti on o f bacteri a l strains, which carried HG2

+

res istance genes, genes for res istance to C D, to luene and naphthalene degradation genes, chlorobiphenyl genes or chlorobenzoate genes. Seri es of re lated genes could be detected when mi xed probes, such as for the different chlorcatecho l, 1,2-dioxygenases are applied . Specific DN A sequences co uld a lso be detected in environmental sources by hybridi zation with probes after amplification of those sequences by using the polymeric chain reaction46. Po lymeric chain reacti on and amplification all owed the detec tio n of specific bacteri al strains at very low levels in samples of various orig ins47. Theoriti cally the use o f conserved DNA sequences in a gene family as Uni versal primers in polymerase chain reactio n amplifi cati on and subsequent cloning o f the ampli fied fragments would a llow the detection and isolation o f a wide variety of genotypes from the enviro nment. Such an approach was used for the characteri zation of vari ations in 16 S rR NA genes from microorganisms in natural communities . Also gene fragments o f the ribulose biphosphate decarboxy lase gene (rbcL) and the nitrogenase gene (ni f H) could be isol ated after polymerase chain reactio n ampli fication o f DNA from environmental samples by using primers for highly

d . . h 4R conserve regIOns 111 t ese genes .

Possible mechanisms and reactions involved The centra l metabo lite of anaerobic degradati on of

aromatic compounds seems to be Co-A thioesters of benzoic ac id or hydroxy ben zoic ac id. Benzene ring undergoes various 'substitutio n' and 'addition ' reactions to fo rm chl oro-, nitro-, methyl substituted aromatic compounds. In order to co mpletely degrade such compounds, the side chains have to be removed first and then the benzene ring is .activated by carboxylation or hydroxylation or Co-A thioesters formation . In the next step, the acti vated ring is converted to a form that can be collected in the central pool o f metaboli sm. The first step depends on nature and complexity of side chains. Various reactions such as dehalogenation , decarboxyl ation , and others are in volved49. The second step is ring fi ss ion or channeling reaction - conversion of different aromatic structures into a few central reacti ve intermediates.

(benzyl Co-A, resorcino l, phlorog lucino l and poss ibly others.

Important novel reactions are : ~ Benzy l Co-A reduction ~ Resorcino l reduction ~ Reducti ve dehydroxylatio n ~ Reducti ve deamination ~ Phenol carboxylation ~ Anilin carboxy latio n ~ Toluene methyl hydroxylation ~ Transhydroxy latio n ~ Reducti ve dehalogenatio n ~ O-demethylati on ~ Anaerobic alpha-oxidatio n ~ Resorc inol hydrati on

New aerobic reactions ./ Hydrol ytic dehalogenati on ./ Mono-oxygenase reductio n

Important enzyme reactions in brief

Decarboxylation Decarboxy latio n plays a rol e in the complete

degradation of the respecti ve aromati c ac ids and in the food chain since they remove the organic ac ids or provide CO2 fo r acetogenesis.

For exampl e, decarboxy lation o f phenyl aceti c ac ids lead to c resols, xy lene or toluene.

Aromatic acids with two or more hydroxy l functions may become decarboxy lated .

O-demethylation, aryl ether cleavage O-methyl ether linkages can be cleaved by an 0-

demethylation reacti on.

Oxidative decarboxylation For example, phenylg lyoxalate is ox idati ve

decarboxylated to g ive (4- hydroxy) benzy l co-A.

Carboxylation Carboxy lation of the aromatic ring is the first step

in degradation of phenol and other pheno li c compounds substituted in ortho position. Pheno l carboxylatio n was studied in nitrate respiring and sulphate reducing bacteri a3

. Enzy me system is referred to as "Pheno l carboxy lases".

Co-enzyme A. thioester formation Benzoic ac id and anal ogues, if they are not

decarboxylated , are converted into their coenzy me. Enzymes for benzoic ac id, 2 amino benzoic ac id and

JOTHIMANI et al.: ANAEROBIC BIODEGRATION OF AROM ATIC COMPOUNDS 1065

others have been purified from Rhodopseudomonas palustris and a denitrifying Pseudomonas sp. The regulation of benzyl co-A ligase has been studued in Rhodopseudomonas3

.

Reductive dehydroxylation This plays an important ro le in the metabo li sm of

phenol, 4 hydroxy benzoate and 4 hydroxy phenyl acetate. The enzy me has been puri fied and it is an Fe­S protein which contains 12 Fe and 12 S!260 Kda and consist of three sub units of 75 , 35 and 17 Kda. This reaction requires a reduced e lectron donor and product is benzy l co-A.

Reductive deamination The reaction plays a ro le in degradati on of aniline

and 4-amino benzoate, the enzy me is acti ve with di fferent reductants. Also it pl ays a role III

degradation of indole and indo lic compounds.

Reductive dehalogenation A sulphate reducing bacterium Desulfomonile

tiedjei able to dechlorinate, 3-chlorobenzoic ac id to benzoic acid with hydrogen or formate as reductant.

Transhydroxylation For example, ga llic acid is first decarboxy lated to

pyrogallol and isomeri zed to phloroglucino l. In thi s reaction, a tetra hydroxy benzene molecule acts as co­substrate.

Aromatic ring hydroxylation Hydroxylation of the aromatic ring may play a

major ro le in the anaerobic metabo lism of toluene in some organisms (under methanogenic and nitrate respiring conditions) .

Nitro group reduction The most common anaerobic process appears to be

the reduction of the nitro substituent via the nitroso and hydroxy lamine compounds to the ammo compound.

Removal of sulpho or sulphonicacid substituents These compounds could serve e ither as sulphur

and/or carbon source. Nothing is known about their anaerobic metabolism.

Benzyl Co-A reduction This intermediate is fo rmed from a large vari ety of

diffe rent compounds, such as phenol, 4-hydroxy­benzoate, P-cresol etc.The reduction of bezoy l Co-A has been demonstrated using a pseudomonas sp.

Phloroglucinol reduction Phloroglucinol an intermediate in the degradation

of a limited number of trihydroxybenzene molecules,

is directly reduced . A soluble oxygen insensitive NADPH dependent phloroglucinol reductase has been purified from Eubacterium oxidoreducens.

Resorcinol reduction Resorcinol is formed some dihydroxybenzoic acids

or form 1,2,4-trihydroxybenzene and is reduced to 1,3-dioxocyclohexane as the ultimate alicyc lic compound in a fermenting clostridium. The reaction is catalysed by a soluble oxygen sensiti ve reductase. The enzyme has not been purified.

In summary, ring reduction and hydrati on in vo lves- the di fferent channeling reactions are combined such that one of several reactive aromatic intermedi ates is obtained . The aromatic ring in thi s compound is acti vated in such a manner that it can be reduced by specific enzy mes. The most general intermedi ate is benzy l Co-A, in which the aromatic ring is acti vated by the thi oesteri fied carboxy l group less common are resorcino l and phlo roglucino l, in which the aromatic ring is acti vated by two of three hydrox yl functions.

Conclusion In the days of accumulating wastes, aromatic

compounds compound the problems of disposal by their recalcitrance, while the other organic wastes can be readily treated by aerobic mechanisms. Decomposi tion of these compounds is comparati vely slower because of the involvement of benzene ring. The energy in volved in the cleavage of these rings is comparati vely hi gher and only a few microbes manage to utili ze them at all. Another di ffi culty is that many of these aromati c compounds reach many anoxic environments where the availability of molecular oxygen is very less. But some of the organi sms have evolved mechani sms to bypass the energy barrier and many of the phenolic compounds are converted to reduced products.

On the application side, many atte mpts have been made to e mploy micro-organi sms wither sing ly or in groups to recti fy contaminated sites. At the outset, the anaerobes play important role in the detoxifying and decomposing many natural and xenobiotic compounds even though the rate at which it is done will have to be improved substantially if we are using these scavengers efficiently . While selection of a natural species is best for release into the environment, a complete deduction of metabo lic paths and regulatory mechanisms will help to make super bugs.

1066 INDIAN J EXP BIOl, SEPTEMBER 2003

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