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Biodegradation of ChlorinatedCompounds—A ReviewPraveena Bhatt a , M. Suresh Kumar a , Sandeep Mudliar a & TapanChakrabarti aa National Environmental Engineering Research Institute (NEERI),Nehrumarg , Nagpur, IndiaPublished online: 23 Nov 2006.

To cite this article: Praveena Bhatt , M. Suresh Kumar , Sandeep Mudliar & Tapan Chakrabarti (2007)Biodegradation of Chlorinated Compounds—A Review, Critical Reviews in Environmental Science andTechnology, 37:2, 165-198, DOI: 10.1080/10643380600776130

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Critical Reviews in Environmental Science and Technology, 37:165–198, 2007Copyright © Taylor & Francis Group, LLCISSN: 1064-3389 print / 1547-6537 onlineDOI: 10.1080/10643380600776130

Biodegradation of ChlorinatedCompounds—A Review

PRAVEENA BHATT, M. SURESH KUMAR, SANDEEP MUDLIAR,and TAPAN CHAKRABARTI

National Environmental Engineering Research Institute (NEERI), Nehrumarg, Nagpur, India

The pressures of an ever-increasing population and industrial de-velopment have led to the addition of an array of man-made chem-icals in the environment, leading to tremendous deterioration inenvironmental quality. Contamination of soil, air, water, and foodis one of the major problems facing the industrialized world to-day. Significant regulatory steps have been taken to eliminate orto reduce production and/or release of these chemicals into theenvironment. A major class of these chemicals is chlorinated com-pounds, most of which are toxic and hazardous. Application ofmicrobial processes to decontaminate environmental media pol-luted with these compounds will require a better understanding ofwhy and how microorganisms can degrade them and utilize themfor their own survival as well as clean the environment. This re-view focuses on different microbial processes for biodegradationof chlorinated compounds and enzymes involved therein that areresponsible for their degradation.

KEY WORDS: aerobic, anaerobic, biodegradation, chlorinatedcompounds, dehalogenation

1. INTRODUCTION

Chlorinated organic molecules constitute the largest single group of com-pounds on the list of priority pollutants compiled by the U.S EnvironmentalProtection Agency (U.S. EPA).1 They are extraneously added into the envi-ronment in large quantities as a result of their widespread use as herbicides,

Address correspondence to Suresh Kumar, National Environmental Engineering ResearchInstitute, Nehrumarg, Nagpur, India. E-mail: drmsureshk@yahoo.com

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insecticides, fungicides, solvents, hydraulic and heat-transfer fluids, plasti-cizers, cleaning agents, fumigants, aerosol propellants, gasoline additives,degreasers, and intermediates for chemical syntheses. The ability of chlori-nated compounds to impart toxicity, bioconcentrate, and persist and con-sequently their ubiquitous distribution in the biosphere has caused publicconcern over their possible effects on the quality of life.2 A list of syn-thetic chlorinated compounds and their use is given in Table 1.3−6 A listof some chlorinated pesticides and their structure is given in Table 2. Somechlorinated compounds also occur naturally in the environment, although inlower concentrations. For example, many different genera of wood rottingfungi produce chlorinated anisyl metabolites in their natural environments.These chloroanisyl-derivative-producing fungi are widespread in nature. Aubiquitous production of chloroanisyl metabolites under natural conditionswas proposed by de Jong et al.7 More than 130 chlorine-containing com-pounds have been isolated from higher plants and ferns. Many compoundsare chlorohydrins, which are isolated along with their related epoxides.8

As is true for many organic compounds, the turnover of chlorinatedmolecules in the environment is largely determined by their susceptibilityto biotransformation by microorganisms.9 Many of the chloro-organics thatare not degraded by bacteria and fungi have the potential to persist in theenvironment and express their toxicity over extended periods of time.10

Thus, identification and application of novel organisms that use chlori-nated pollutants for growth have become an important area of research today.Further, process optimization for biodegradation of these hazardous chemi-cals requires an understanding of microorganisms involved in the degrada-tion, their nutrient requirements, the biochemistry of their mediated reactions,and why they promote these reactions.

2. UTILIZATION OF CHLORINATED COMPOUNDSBY MICROORGANISMS

The biological destruction of toxic and hazardous chemicals is also based onthe principles that support all ecosystems. These principles involve the circu-lation, transformation, assimilation of energy and matter.11 Microorganismsconvert complex organic compounds, via their central metabolic routes, toCO2 or other simple organic compounds. The oxidation yields energy andreducing equivalents that are used for conversion of a part of the intermedi-ates to cell mass (assimilation), enabling growth of the organisms that carryout the degradation process.12

Degradation of compounds of natural origin is usually easy to achieve,and organisms that bring about their degradation can be easily isolated fromtheir natural environments. However, in general, compounds having a struc-ture that is different from naturally occurring compounds (xenobiotics, most

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Biodegradation of Chlorinated Compounds 167

TABLE 1. Major Chlorinated Hydrocarbons (HC) and Their Applications

Chlorinated HC Major uses

ChloromethanesMonochloromethane Production of silicones, tetramethyllead, methylcellu-

lose, other methylation reactions3

Dichloromethane Degreasing agent, paint remover, pressure mediator inaerosols; extract technology3

Trichloromethane Production of monochlorodifluoromethane (for the pro-duction of tetrafluoroethene, which is used for themanufacture of Hostaflon and Teflon), extractant forpharmaceutical products3

ChloroethanesMonochloroethane Production of tetraethyllead, production of ethylcellu-

lose; ethylating agent for fine chemical production,solvent for extracting processes3

1,1- Dichloroethane Feedstock for the production of 1,1,1-trichloroethane3

1,2-Dichloroethane Production of vinyl chloride, production of chlorinatedsolvents such as 1,1,1-trichloroethane and tri- andtetrachloroethane, synthesis of diethylenediamines3

1,1,1-Trichloroethane Dry cleaning, vapor degreasing, solvent for adhesivesand metal cutting fluids; textile processing3

1,1,2-Trichloroethane Intermediate for production of 1,1,1-trichloroethaneand 1,1-dichloroethane3

ChloroethenesMonochloroethene Production of polyvinyl chloride (PVC), production of

(vinyl chloride) chlorinated solvents, primarily 1,1,1-trichloroethane4

Trichloroethene Solvent for vapor degreasing in the metal industryand for dry cleaning, extraction solvent, solvents informulations for rubbers, elastomers and industrialpaints4

Tetrachloroethene Solvent for dry cleaning, metal degreasing, textile fin-ishing, dyeing, extraction processes, intermediatefor the production of trichloroacetic acid and somefluorocarbons4

2-Chloro-1,2-butadiene Starting monomer for polychloroprene rubber4

(Chloroprene)Chlorinated paraffins Plasticizers in PVC; flameproofing agents in rubber tex-

tiles, plastics, H2O repellent and not—preventiveagents; elastic sealing compounds paints & var-nishes; metalworking agents (cutting oils); leatherfinishing4

Chlorinated aromatic HCMonochlorobenzene Production of nitrophenol, nitoranisole, chloroaniline,

phenylenediamine for the manufacture of dyes, cropprotection products, pharmaceuticals and rubberchemicals4

1,2-Dichlorobenzene Production of 1,2-dichloro-4-nitrobenzene for the pro-duction of dyes and pesticides; production of disin-fectants, room deodorants4

1,4-Dichlorobenzene Production of disinfectants, room deodorants, mothcontrol agent; production of insecticides; produc-tion of 2,5-dichloronitrobenzene for the manufactureof dyes, production of polyphenylenesulfide-basedplastics4

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TABLE 1. Major Chlorinated Hydrocarbons (HC) and Their Applications (Continued)

Chlorinated HC Major uses

Chlorinated toluenes Hydrolysis of cresol, solvent for dyes; precursors fordyes, pharmaceuticals, pesticides, preservatives anddisinfectants4

Chlorophenols Preparation of agricultural chemical (herbicides etc)5

Chlorophenonyalkanoicacids

Herbicides6

Side-chain chlorinatedaromatic HC

Chloromethylbenzene(benzylchloride)

Production of plasticizer, benzyl alcohol, phenylaceticacid, quarternary ammonium salts, benzyl esters, triph-enylmethane dyes, dibenzyl disulfide, benzylphenol,benzylamines3

Dichloromethyebenzene(benzalchloride)

production of benzaldehyde3

Trichloromethylbenzene(benzotrichloride)

Production of benzoylchloride; Production of pesticides; UVstabilizers and dyes3

Pesticides, herbicidesand fungicides

For seed treatment, for treatment of diseases of plants,animals, and humans

of which are toxic and hazardous) are more difficult to degrade.10 Never-theless, in the recent past, an array of microorganisms has been identifiedthat use xenobiotics such as chlorinated alkanes, chlorinated halohydrins,polychlorinated biphenyls, and chlorobenzenes for their survival.

2.1. Energy Metabolism

Several bacterial strains have been isolated that utilize chlorinated com-pounds for synthesis of energy. Many have been shown to couple reductivedechorination to energy metabolism.13−15 Desulfomonile tiedjei uses H2 orformate as an electron donor and 3-chlorobenzoate as a terminal electronacceptor in a respiratory process.16−18 Chemo-osmotic coupling of reductivedechlorination and ATP synthesis has been demonstrated in bacterium DCB-1.18 This organism can biosynthesize ATP by coupling hydrogen oxidation toreduction of the C–Cl bond of 3-chlorobenzoate. Using acetate or fumarateas electron donor, the isolate CP-1 grows via reductive dechlorination ofchlorophenol (CP).19 Christof Holliger et al.20 have studied a highly purifiedenrichment culture that couples dechlorination of tetrachloroethene (TeCE)to growth. They demonstrated that PER-K23 catalyzes transformation of PCE(perchloroethene) via TCE (trichloroethene) to cis-1,2 DCE (dichloroethene)and synthesizes energy via electron transport phosphorylation.20 Bradley andFrancis21 studied aerobic microbial mineralization of DCE as sole carbon sub-strate. Methylobacterium, Methylophilus, and Hyphomicrobium are aerobicbacteria capable of growth with DCM (dichloromethane) as sole source of

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Biodegradation of Chlorinated Compounds 169

TABLE 2. Some Chlorinated Pesticides and Their Structures

Number Pesticide Structure

1 DDT: 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane

2 2,4-D: (2,4-dichlrophenoxy)acetic acid

3 2,4,5-T: (2,4,5-trichlorophenoxy)aceticacid

4 Dioxin: 2,3,7,8-tetrachlorodibenzo-p-dioxin

5 Chloronitrofen: (4-nitrophenyl-2,4,6-trichlorophenyl ether)

6 Lindane: V-hexachlorocyclohexane

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TABLE 2. Some Chlorinated Pesticides and Their Structures (Continued)

Number Pesticide Structure

Aldrin: (1,2,3,4,10,10-hexachloro-1,4,5,6,7,8-octahydro-1,4-endo,exo-5,8-dimethanonaphthalene

8 Dieldrin: (1,2,3,4,10,10-hexachloro-6,6-epoxy-1,4,5,6,7,8-octahydro-1,4-endo, exo-5,8-dimethanonaphthalene

9 Heptachlor: (1,4,5,6,7,8-heptachloro-3,5,7-tetra-hydro-4,7-methanoindene

10 Chlordane

11 Dicamba: (3,6-dichloro-2-methoxybenzoate)

12 PCP

13 TPN: 2,4,5,6-(tetrachloroisophthalonitrile)

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Biodegradation of Chlorinated Compounds 171

TABLE 2. Some Chlorinated Pesticides and Their Structures (Continued)

Number Pesticide Structure

14 Diuron: 3-(3,4-dichlorophenyl)-1,1-dimethylurea

15 Benthiocarb: thiobencarb, 5-4-chlorobenzyl-N, N-di-ethylcarbamate

16 Techlofthalam: N-(2,3dichlorophenyl)-3,4,5,6-tetra-chlorophthamic acid

17 Nitrapyrin: 2-chloro-6-trichloromethylpyridine

18 Picloram: 4-amino-2 carboxy-3,5,6-trichloropyridine

19 Murin: dodecachlorooctahydro-1,3,4-methano-2H -cyclobuta(cd )pentalene

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energy and carbon.22 Susanna et al.23 have demonstrated DCM as sole “C”source for an acetogenic mixed culture.

Desulfitobacterium chlororespirans gains energy from the reduc-tive ortho-dechlorination of 3-chloro-4-hydroxy benzoate and 2,3-di- andpolychloro-substituted phenols.24 Neumann et al.25 unambiguously demon-strated coupling of reductive dechlorination to respiratory growth in Desul-fitobacterium multivorans.

However, utilization of chlorinated compounds is not based purely onenergy metabolism. Although traditionally it was believed that organismsmust obtain energy from an organic compound by degrading it, now it hasbeen shown that organisms growing at the expense of one substrate canalso transform a different substrate that is not associated with that organismsenergy production, “C” assimilation, or any other growth process. This modeof activity is called “cometabolism.”11

2.2. Cometabolism

Cometabolism is defined as the degradation of a compound only in the pres-ence of another organic material that serves as the primary energy source.26

A number of laboratory studies have demonstrated that several chlorinatedhydrocarbons are transformed cometabolically by bacteria that degrade thechlorine unsubstituted aliphatic and/or aromatic hydrocarbons.27,28

Several studies on chlorinated solvents undergoing fortuitous dechlo-rination by microorganisms growing on other electron donors and accep-tors have also been documented.29 Nitrosomonas europea can cometabo-lize dichloromethane (DCM), trichloromethane (TCM), 1,1,2-trichloroethane,1,1,1-trichloroethane, and 1,2,3-trichloropropane while utilizing ammoniaas the primary substrate.30 Several bacteria capable of oxidizing toluene,methane, and ammonia can cometabolize TCE, DCE, and vinyl chloride(VC).31 Pseudomonas cepacia G4 is one such organism that uses toluene andcan degrade TCE cometabolically.32 Reductive dechlorination or reduction ofTeCM (tetrachloromethane) by Escherichia coli K12 under fumarate respir-ing conditions and by a denitrifying strain Pseudomonas KC are cometabolicprocesses that are mediated by electron carriers of the respiratory electrontransport chain.33,34

It has been observed that in some bacteria if nonhalogenated diphenyl-methane is added as a primary substrate, the chlorinated substituted formis degraded by cometabolism.26 Hage et al.35 have reported Pseudomonasstrain DCA1 could cometabolize a broad range of chlorinated methanes,ethanes, propanes, and ethenes using chloroacetic acid as cosubstrate.Phenol-oxidizing microorganisms have been shown to effectively transformcis- and trans-DCE and TCE in laboratory as well as in situ field studies.36 Al-caligenes denitrificans and Rhodococcus erythropolis can cometabolize TCE,DCE, and VC. A Xanthobacter has been reported to degrade TCE, VC, cis- andtrans-1,2-DCE, 1,3-DCP (dichlorophenol), and 2,3-DCP cometabolically.37

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Biodegradation of Chlorinated Compounds 173

The phenomenon of cometabolism has been attributed to the pro-duction of broad-specificity enzymes. Both the primary substrate and thechlorinated compound compete for the same enzyme.26 It has been re-ported that several oxygen-catalyzed dehalogenation reactions of chlorinatedmethanes, ethanes, and ethylenes are due to multifunctional enzymes withbroad specificity or involve enzymes from aromatic degradative pathways.6

For a cometabolic mode, the degradation rate of the target chlorinated com-pound is dependent on the electron flow from the primary substrate.

3. THERMODYNAMICS AND KINETICS OF BIODEGRADATION

Principally microorganisms couple only those half-reactions that yield themaximum free energy. Reduction of a chlorinated compound is an exother-mic reaction, with the energy released from the reaction comparable to nitratereduction and much higher than either methanogenisis or sulfate reductionunder identical physiological conditions.38 During energy metabolism, en-ergy available from all reductive dechlorination reactions is of a similar or-der of magnitude, irrespective of the parent compound and the number orposition of chlorines, since most of the energy becomes available due to thechange in the oxidation state of chlorine (Cl+ or Cl−).38 Thus, free energyfrom each chlorine atom removed for a host of chlorinated organics has beencalculated to vary between −130 and −171 kJ/Cl atom removed.39

Holliger et al.20 showed that the reductive dechlorination of PCE tocis-1,2-DCE by PER-K23 is an exergonic reaction with a Gibbs free energychange of −377.5 KJ/mol PCE. They suggested that this energy is enough tosynthesize approximately 5 mol of ATP, assuming that an energy differenceof 70 kJ is needed for the formation of 1 mol of ATP in an irreversible reactionunder identical physiological conditions. According to Hollinger et al., sinceATP formation by substrate level phosphorylation is unlikely to occur uponH2 oxidation during dechlorination, electron transport phosphorylation mightbe the mechanism of ATP synthesis.20

Metabolic rates in general are several orders of magnitude higherthan cometabolic rates.40 The cometabolic rates of PCE dechlorination byMethanosarcina sp. and Acetobacterium woodii were 3.5 × 10−5 µmol/h/mgprotein and 3.6 × 10−3 µmol/h/mg protein, respectively, in contrast to ametabolic PCE dechlorination rate of 3 µmol/h/mg proteint by Dehalospir-illum multivorans.14 Similarily, Futamata et al.41 have shown that Vmax/K s

values for phenol degradation were three orders of magnitude higher thanthe values for TCE in several phenol-oxidizing bacteria. Futamata41 and hiscolleagues concluded that a general relationship exists between reductionpotential, chlorine substituent number, chlorine substitution pattern, anddechlorination rate of chlorinated aliphatic compounds.

Vogel et al.28 have suggested that carbon–chlorine bond reductivedechlorination cleavage rates may be proportional to the heat of formation

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of a resultant organic radical following one electron input or may cor-relate with the standard reduction potential of the chlorinated aliphaticcompound.

4. BIODEGRADATION OF CHLORINATED COMPOUNDS

Biodegradation of chlorinated compounds follows two pathways namely“aerobic degradation” or “anaerobic degradation.” However, irrespective ofthe pathway followed, the extent of degradation depends on the struc-ture of the compound, the number of chlorine substituents, and the posi-tion of chlorine in the molecules. Depending on the structure, chlorinatedcompounds can be either oxidized or reduced. Reduction is possible be-cause of their electronegative character, which makes them highly electrondeficient.26

4.1. Aerobic Degradation

During aerobic degradation of chlorinated compounds by microorganisms,molecular oxygen serves as the electron acceptor. Several chloroaliphaticcompounds have been shown to degrade aerobically. A number of stud-ies have demonstrated that microorganisms degrade DCE under aerobicconditions.21,42−44 Seung-Bong Lee et al.45 observed sustained degradationof TCE in a suspended growth reactor by an Actinomycetes enrichment cul-ture. Aerobic mineralization has been well documented for chlorobenzeneswith up to four chlorine substituents in microcosms and by pure cultures.46−54

Several of the chlorobenzenes (containing one, two, three, or four chlorinesubstituents) could be biotransformed only under aerobic conditions andwere unstable in the absence of molecular oxygen.53 It has been reportedthat 4-chlorophenol (4-CP) can be partially or completely degraded aerobi-cally by several bacteria, including Pseudomonas,55 Alcaligenes,56 Rhodococ-cus, Azotobacter58 etc. Richard and Michael59 studied degradation of pen-tachlorophenol (PCP) by Phanerochaete spp. and studied its sensitivity tothe compound. A list of microorganisms degrading some aliphatic chlori-nated compounds aerobically is given in Table 3.60−64

TABLE 3. Aerobic Degradation of Some Chlorinated Aliphatics

Number Compound Microorganism References

1 1,2-Dichloroethane X. autotrophicus 60, 612 1,1,2-trichloroethane P. putida 623 Trichloroethylene Methylomonas methanica 634 CCl4 Escherichia coli K-12 64

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Biodegradation of Chlorinated Compounds 175

4.2. Anaerobic Degradation

In the anaerobic mode of degradation the electron acceptor is a moleculeother than O2. This could be NO−

3 , SO2−4 , Fe3+, H+, S, fumarate, trimethy-

lamine oxide, an organic compound, or CO211. The term “dehalorespiration”

has been coined for anaerobic bacteria that couple the reductive dehalo-genation of chlorinated aliphatic and aromatic compounds to ATP synthesisvia an electron transport chain.15 Reductive dechlorination or reductive de-hydrogenolysis is a common biotransformation pathway for chloroaliphaticscontaining one or two carbon atoms, under methanogenic conditions.29 ChunChun et al.65 studied the biotic transformation of TeCE under methanogenicconditions. A strictly anaerobic homoacetogenic bacterium and an uncharac-terized anaerobic mixed culture were shown to use chloromethane as a ‘C’and energy source.23

Most of the chlorinated aromatic compounds and several pesticides areknown to be best degraded under anaerobic conditions. Ramanand et al.66

have reported rapid degradation of chlorinated benzenes and toluenesunder methanogenic conditions. Several chlorinated aromatic compoundshave been shown to be degraded under methanogenic conditions. These in-clude 2,4,5-trichlorophenoxyacetate, 3-chlorobenzoate, 2,4-dichlorophenol,4-chlorophenol, 2,3,6-trichlorobenzoate, and dichlorobenzoates.67,68

Jiangzhong He et al.69 have reported complete detoxification of VC byan anaerobic enrichment culture, which they later identified as Dehalo-coccoides sp. Buser and Muller70 have studied degradation of pesticidehexachlorocyclohexane (HCH) and its isomers in sewage sludge underanaerobic conditions. Studies by Tina Holscher et al.71 showed anaerobicreductive dechlorination of chlorobenzene congeners in cell extracts ofDehalococcoides strain CBDB1. Chlorophenol degradation coupled toSO2−

4 reduction has been documented by Haggblom and Young.72 Theysuggested that degradation of chlorinated aromatic compounds not onlytakes place under sulfate-reducing conditions but is in fact coupled to sulfatereduction.62 Vargas et al.73 have given an account of anaerobic dechlorina-tion of chlorinated dioxins in estuarine sediments. A list of microorganismsdegrading some aliphatic chlorinated compounds anaerobically is given inTable 4.74−80

4.3. Sequential Degradation

Although degradation of chlorinated aliphatic and aromatic compounds hasbeen reported both under aerobic and anaerobic conditions, sequential useof these processes always has an advantage over using them individually forcomplete mineralization of heavily chlorinated compounds. It is generallyimplied that aerobic microbes often fail to metabolize heavily chlorinatedcompounds. For example, several bacteria capable of oxidizing TCE, DCE,

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TABLE 4. Anaerobic Degradation of Some Chlorinated Aliphatics

Number Compound Microorganism References

1 Carbon tetrachloride Methanogenic enrichment 74Clostridium sp. 75

2 1,1,1-Trichloroethane(TCE)

Sulfate reducing enrichment,methanogenic enrichment

76

3 Vinyl chloride Anaerobic mixed culture 774 Dichloromethane (DCM) Anaerobic consortia 785 Trichloroethylene Anaerobic consortia 79, 80

and VC by using nonspecific enzymes cannot oxidize TeCE by any of theseenzyme systems.81−84 Aerobic bacteria that rapidly biodegrade monochlori-nated benzenes are usually unable to degrade heavily chlorinated benzenecompounds.68 Similarly, increased resistance of chloroalkenes to biologicalreductive dechlorination has been observed in anaerobic reactors and anaer-obic freshwater microcosms.85,86

Therefore, it has been suggested that detoxification and completemineralization of chlorinated wastes can be easily achieved by usinga sequential treatment process, that is, anaerobic followed by aero-bic treatment. For instance, the fungicide HCB (hexachlorobenzene) andpolychlorinated biphenyl (PCB) undergo reductive dechlorination in anaero-bic environments.83,84,87 The products are congeners bearing fewer chlorinesubstituents, which are more susceptible to biodegradation by aerobicbacteria.52,88 A sequential treatment step will ensure total mineralization ofthese chlorinated toxic compounds.

4.4. Role of Electron Donors in Dechlorination

Reductive dehalogenation reaction, whether catalyzed by a transition metal,bacterial cofactors, or an enzyme, requires two electrons. Therefore, a sourceof electrons must be available for the reaction to take place.38 The sourceof electrons (or electron donor) for a dechlorination reaction is usually areduced substrate provided for microbial growth. Nies and Timothy89 studiedthe effects of different organic substrates on the ability of anaerobic sedimentenrichment to reductively dechlorinate polychlorinated biphenyls. They usedacetate, acetone, methanol, and glucose and found that the relative rates ofdechlorination were in the order methanol > glucose > acetone > acetatefed cultures.89

De Bruin et al.90 observed biological reductive dechlorination of TeCEto ethane with lactate as the electron donor. Gibson67 observed that addi-tion of butyrate, propionate, ethanol, or acetate increased not only the rateof dehalogenation of trichlorophenoxyacetic acid but also the extent of itsdegradation. Hydrogen, formate, ethanol, propionate, or acetate can serve as

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Biodegradation of Chlorinated Compounds 177

the source of reducing equivalents required for dechlorination in the bacteriaDesulomonile tiedje.17,18

A similar observation was seen in case of trichlorophenol (TCP) degra-dation, where yeast extract was the preferred primary substrate and resultedin complete degradation of the target compound within 3 days.91 With pep-tone and casamino acid, complete transformation was observed only after6–7 days.75 Studies by Holliger et al.92 showed that HCB, all three isomers ofTeCE, 1,2,3-TCB (trichlorobenzene), and 1,2,4-TCB were reductively dechlo-rinated by enrichment culture in the presence of lactate, glucose, ethanol, orisopropanol as electron donors. Lactate, ethanol, and H2 appeared to be thebest substrates. Moreover, dechlorinating activity could not be maintainedwhen electron donor was not added.92

Gibson and Sewell93 observed that lactate, propionate, crotonate,butyrate, and ethanol stimulated dechlorination activity of TeCE inmethanogenic slurries made with aquifer solids. Acetate, methanol, andisopropanol did not support dehalogenation.93 For bacteria like the Nitro-somonas sp., capable of degrading several chlorinated aliphatic compounds,ammonia serves as the electron donor. A study demonstrated that dehalo-genation of DCE in a contaminated soil requires fatty acids and alcohols aselectron donors. Supporting evidence was also given to show that the dechlo-rination process stops once the electron donor is depleted.94 Smatlak et al.95

observed that PCE dechlorination rates decreased significantly at lower H2

concentrations, which was added as an electron donor in the experiment.Dechorination of PCP was enhanced by the addition of glucose to a UASBreactor fed with PCP and phenol.96

4.5. Role of Electron Acceptors in Dechlorination

All energy-yielding reactions are oxidation–reduction reactions. The reduc-tion reaction, that is, the reaction involving the electron acceptor, establishesthe metabolism mode.26 Microbes preferentially utilize electron acceptorsthat provide the maximum free energy during respiration.97 Among the com-mon electron acceptors used by microorganisms, O2 typically provides themaximum free energy during electron transfer, followed by nitrate, Mn(IV),Fe(III), SO2−

4 , and CO2.98

Cobb and Bouwer98 used a mixture of primary electron acceptors likeO2, nitrate, and sulfate for the transformation of 1,1,1-TCE, TeCE, and chlori-nated benzenes, and suggested sulfate to be an important primary acceptor.Experimental studies with a biofilm using a single electron acceptor showedthat halogenated aliphatic compounds such as TCE, chloroform, and otherscould be transformed under methanogenic and sulfate reducing conditions.85

Chlorinated compounds are stronger oxidants than nitrate.28 On the ba-sis of such thermodynamic considerations, chlorinated hydrocarbons havebeen shown to act as terminal electron acceptors in a respiratory process.39,99

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Cupples100 observed growth of a Halococcoides-like organism on VC and cis-dichloroethene as electron acceptor. Dehalococcoides ethenogens strain 195completely dechlorinates PCE to ethene using H2 as electron donor and PCEas the electron acceptor.101 A study by Holliger et al.20 revealed that a highlypurified enrichment culture could use only PCE or TCE as electron acceptorand O2, NO−

3 , NO−2 , SO2−

4 , SO2−3 , S2O

2−3 , S, or CO2 could not replace PCE

or TCE as electron acceptor. Even organic electron acceptors such as ace-toin, acetol, dimethyl sulfoxide, fumarate, and trimethylamine N -oxide werenot utilized by the organisms.20 PCE as an electron acceptor was used byan acetate-oxidizing anaerobic bacteria identified as Desulfomonas michiga-nensis sp.nov.102

4.6. Role of Transition Metal Cofactors in Dechlorination

Transition metal cofactors can mediate nonspecific reactions with hydropho-bic chlorinated pollutants that gain entry into bacterial cells by partitioningthrough membranes.103 There are two different classes of transition metalcofactors found in bacteria that grow under anaerobic conditions.104 In thefirst type, the metal is coordinated by a stable macrocyclic ligand system,which in turn can be bound by proteins. In the second type, metal(s) is (are)directly coordinated to protein ligands. Both type of redox-active centersdisplay great versatility in their biological functions.103

The cobalt-containing cobalamins and the iron coenzyme hematin(II)show catalytic activities in addition to their biological role as electroncarriers.105,106 Iron–S clusters, which also function in electron transfer, arenow implicated as key participants in several enzyme-catalyzed hydrolyticreactions.107 Gantzer and Wackett103 noted that bacterial transition metalcoenzymes vitamin B12 (Co), coenzyme F430 (Ni), and hematin (Fe) cat-alyzed the reductive dechlorination of polychlorinated ethylenes and ben-zenes, whereas the electron-transfer proteins four-iron ferridoxin, two-ironferrodoxin, and azurin (Cu) did not. Cobalamins, coezyme F430, and hematinhave recently been shown to dehalogenate chlorinated methanes in the pres-ence of a reductant.107 Carbon tetrachloride (CT) degradation rates increaselinearly with higher intracellular vitamin B12 content.108 In many cases, themicrobial transformation of CT is considered to be closely related to thepresence of microbial cofactors such as porphinoids and corrinoids.94 Corri-noids such as aquocobalamin or methylcobalamin catalyze the reduction oftetrachloromethane or trichloromonofluoromethane with titanium(III) citrateor with dithiothreitol as electron donors.109 Klecka and Gonsior110 observedtransformation of CT, chloroform, and 1,1,1-tetrachloroethane by iron por-phyrins with sulfide as the reductant. More recently, zero-valent iron has alsobeen reported to catalyze reductive dechlorination reactions at extremelyhigh rates.111

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Biodegradation of Chlorinated Compounds 179

5. ENZYMES INVOLVED IN DECHLORINATION

Microorganisms have evolved a diverse potential to transform and degradehalogenated organic compounds. They produce an array of enzymes thatbring about dehalogenation and degradation of both aliphatic and chloroaro-matic compounds. The reactions catalyzed by such enzymes can be broadlyclassified as follows:

Reaction Enzymes

Oxidative dehalogenation Mono- or dioxygenasesDehydrohalogenation DehydrohalogenasesSubstitutive dehalogenation HalidohydrolasesDechlorination via methyl transfer MethyltransferasesReductive dehalogenation Dehydrohalogenases

5.1 Oxidative Dehalogenation

Oxidative dechlorination of aliphatic chlorinated compounds is a result ofmono- or dioxygenase enzymes that function via metabolic or cometabolicreactions. The chlorinated hydrocarbon competes along with the growth sub-strate of the organism for the active site of the oxygenase enzyme. The organ-isms, however, are not known to benefit from the cometabolic processes.40

The initial step in the aerobic transformation of chlorinated alkenesis generally assumed to be an epoxidation of the carbon–carbon doublebond.112 The subsequent metabolism of the reactive haloepoxides is notknown in detail, but extensive dehalogenation is frequently observed.110

An example of this kind of dehalogenation is by methane monooxygenase(MMO), which is thought to catalyze the conversion of haloalkenes such asTCE to its epoxide, which subsequently undergoes isomerization or hydrol-ysis. The reaction is represented by40:

where (a) is trichloroethene, (b) methane monooxygenase (MMO), and (c)epoxide.

A high degree of specificity of this enzyme toward TCE was observedin Methylosinus trichosporium OB3b.2,113 There probably are differentmechanisms of TCE oxidation by oxygenases. Microbial oxidation of TCEhas been reported to be catalyzed by toluene 2,3-dioxygenase,114−116

toluene2-monooxygenase,117−120 toluene 4-monooxygenase,121 phe-nol hydroxylase,122 and 2,4-dichlorophenol hydroxylase and propane

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monooxygenase,123 and the involvement of separate dioxygenases wasnoted from data on plasmid profiles and DNA hybridization in Pseu-domonas putida,111 which utilizes a broad range of mono-, di-, andtrichlorinated benzoates.124 Similar to methane monooxygenase, ammonia-monooxygenase oxidizes not only TCE but a variety of n-chlorinatedalkenes.6 Oxygenolytic dehalogenation of haloaromatic compounds is eithercatalyzed by specific oxygenases or occurs during a conversion, catalyzedby the enzyme for the corresponding unsubstituted substrate.

Two-component dioxygenases such as 4-chlorophenyl acetate, 3,4-dioxygenase, and 2-halobenzoate 1,2-dioxygenase preferentially cat-alyze chloroaromatic compounds.125,126 In the degradation of 1,2,4,5-tetrachlorobenzene by Pseudomonas strain PS14, an initial 5,6-dioxygenatingattack is followed by spontaneous elimination of HCl during rearomatizationof the dehydrodiol, yielding 3,4,6-trichlorocatechol.127

Dioxygenolytic dechlorination of 2,2′-dichlorobiphenyl, 2,3′-dichlorobiphenyl, and 2,5,2′-trichlorobiphenyl at the ortho position iscatalyzed by biphenyl 2,3-dioxygenase of Pseudomonas strain LB400.128

All these dioxygenases have been proposed to catalyze the formation ofcis-diols, which spontaneously rearomatize with concomitant Cl2 elimi-nation, yielding a catechol product. In the first step of PCP degradationby Sphingomonas chlorophenolica ATCC 39723, a soluble flavoproteinmonooxygenase catalyzes its NADPH-dependent conversion to tetrachloro-p-hydroquinone.129,130

5.2. Dehydrohalogenation

This type of dechlorinating process eliminates HCl from the haloor-ganic substrate, leading to the formation of a double bond. Dehy-drohalogenation occurs during the mineralization of insecticide γ -HCHby Sphingomonas paucimobilis UT26.131 The elimination of HCl fromboth γ -HCH and an intermediate metabolite γ -pentachlorocyclohexeneis catalyzed by a dehydrochlorinase designated LinA.132 The enzymecatalyzes the release of three chloride ions per molecule of γ -HCH,but its substrate specificity is narrow. α-HCH, γ -HCH, δHCH, α-pentachlorocyclohexene, and γ -pentachlorocyclohexene are the only sub-strates converted. It has been suggested that Lin A catalyzes the stere-oselective dehydrochlorination of HCH with a trans and diaxial pairof hydrogen and chloride. Two other dehydrochlorinase enzymes havealso been described, namely, glutathione-dependent DDT dehydrochlori-nase from the housefly and the 3-chloro-D-alanine dehydrochlorinase fromP. putida, which requires pyridoxal 5′-phosphate.131 Dehydrohalogenasesare also involved in the ortho cleavage of chlorocatechols, which re-sults in chlorinated cis-muconates, which are cycloisomerized to dienelactones.133

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Biodegradation of Chlorinated Compounds 181

5.3. Substitutive Dehalogenation

Substitutive dehalogenation of chlorinated compounds takes place by threedifferent processes:

1. Hydrolytic processes.2. Thiolytic processes.3. Intramolecular substitution reactions.

5.3.1 HYDROLYTIC PROCESSES

Hydrolytic dehalogenation of several heterocylic, aromatic, and aliphaticcompounds has been reported.2,134−138 These reactions are catalyzed by hali-dohydrolases.

Hydrolytic dechlorination of haloalkanes was first found with thehaloalkane dehalogenase from the nitrogen-fixing hydrogen bacterium Xan-thobacter autotrophicus GJ10. Because of the presence of two halidohydro-lases, strain GJ10 is capable of rapid utilization of 1,2-dichloroethane. Boththese dehalogenases in X. autotrophicus are synthesized constitutively.139

These enzymes have a broad specificity and catalyze the dehalogentationof more than 24 haloaliphatic compounds. The haloalkane dehalogenasegene dhl A has been cloned and sequenced.140 One haloalkane halidohy-drolase encoding gene is present in the plasmid (designated pXAU1), whilethe gene encoding the second enzyme, 2-haloalkanoic acid halidohydrolase,is located on the chromosome. Nucleophilic displacement with H2O wassuggested as the mechanism of halide release.141 Asp-24 is the nucleophilicresidue attacking the substrate. It is assumed that the covalent intermediateis an ester, which must be subsequently cleared by water molecule, releasingthe alcohol.142 The hydrolytic dechlorination reaction of 1,2-dichloroethanein Xanthobacter autotrophicus GJ10 is given as follows40:

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(I) 1,2-Dichloroethane; DhlA, haloalkane dehalogenase.(II) 2-Chloroethanol; MoX, alcohol dehydrogenase.

(III) Chloroacetaldehyde; Ald, aldehyde dehydrogenase.(IV) Chloroacetate; Dhl B, 2-haloacid dehalogenase.(V) Glycolate; PQQ, pyrroquinoline quinone.

Since delocalization of the pi electrons considerably stabilizes the aro-matic ring system, it was previously thought unlikely that bacteria haveevolved enzymes for the direct hydrolysis of the aromatic carbon–halogenbond. Deethylsimazine, a monohydroxylated s-triazine derivative, has con-siderable aromatic character, but in contrast to the benzenoid ring, delocal-ization of the pi electrons is not complete. Hydrolytic removal of substituentshas been described for various s-triazines.143 Cook and Hutter144 have shownthat two isofunctional but different enzyme fractions from Rhodococcuscorallinus NRRLB-15444R hydrolytically dechlorinated diethylsimazine to N -ethylamine. No cofactors were required for dechlorination. This hydrolyticsubstitution at the aromatic ring is chemically feasible because of the lowelectron density at the ring carbon atoms.144

An example for hydrolytic dehalogenation reaction is the conversionof 4-chlorobenzoate to 4-hydroxybenzoate. This reaction requires three en-zymes, namely 4-chlorobenzoate coenzyme A (CoA) ligase, 4-chlorobenzoyl-CoA dehalogenase, and 4-hydroxybenzoyl CoA thioesterase. This conversionhas been shown to be catalyzed by a number of bacterial strain belongingto the genera Pseudomonas, Arthrobacter, Acinetobacter, Alcaligenes, No-cardia, and Corynebacterum (Dunaway Mariano and Babitt145). In the con-version of 4-chlorobenzoate to 4-hydroxybenzoate by Pseudomonas strainCBS3, Loffler and Muller146 identified 4-chlorobenzoyl CoA as an intermedi-ate in the dehalogenation reaction and proposed the reaction mechanism.In the first step, a 4-chlorobenzoate lyase catalyzes the adenylation of thecarboxy group followed by displacement of the AMP, a thiol group fromCoA, leading to the formation of the thioester 4-chlorobenzoyl CoA. Theformation of the CoA ester activates the substituent in the para position fora nucleophilic attack and enables the substitution of the chlorine by a hy-droxyl group from H2O, catalyzed by dehalogenase.146 The reaction can berepresented as2:

where (I) is 4-chlorobenzoate CoA ligase, (II) is 4-chlorobenzoyl CoA de-halogenase, and (III) is 4-hydroxybenzoyl CoA thioesterase.

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Biodegradation of Chlorinated Compounds 183

5.3.2. THIOLYTIC PROCESSES

The thiolytic substitutive dehalogenation process is catalyzed by glutathioneS-transferase enzymes. This process has been extensively studied in methy-lotrophic bacteria. Dechlorination of dichloromethane by facultative methy-lotrophic bacteria is catalyzed by inducible glutathione S-transferases.Dichloromethane is converted to formaldehyde and inorganic chloride withS-chloromethylgutathione as intermediate and the formaldehyde so formedis a central metabolite of methylotrophic growth.40 Pseudomonas strains, Hy-phomicrobium strains, and several Methylobacterium sp. strains have beenshown to contain these enzymes.146−149 The following reaction has beentaken from ref. 40:

where (I) is dichloromethane dehalogenase, (II) is formaldehyde dehaloge-nase, and (III) is formate dehydrogenase.

5.3.3. INTRAMOLECULAR SUBSTITUTION REACTIONS

These reactions are catalyzed by halohydrin–hydrogen halide lyases, alsocalled halohydrin epoxidases. They were first discovered by Castro150 andBartnicki151 in 1968 from a 2,3-dibromo-1-propanol utilizing Flavobacteriumsp. They constitute a unique group of dehalogenating enzymes.150,151

In 1989, Van den Wijngaard et al.152 reported the degradation ofepichlorohydrin and halohydrins by Pseudomonas strain AD1, Arthrobacterstrain AD2, and Coryneform strain AD3. Halohydrin dehalogenase from strainAD2 converted C-2 and C-3 chloroalcohols and was active with chloroace-tone and 1,3-dichloroacetone as well, yielding epoxides as products. Neithercofactors nor O2 was required for the dehalogenation. Thus, the reactionmechanism was thought to proceed via intramolecular substitution.152,153 Thereaction did not require any cosubstrate, and purified haloalcohol dehaloge-nase from AD2 showed no immunological cross-reactions with haloalkaneor 2-haloacid halidohydrolases.154

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5.4. Dechlorination via Methyl Transfer

A chloromethane dehalogenase, which is inducible by chloromethane, trans-fers the methyl group of its substrate onto tetrahydrofolate, producing methyl-tetrahydrofolate and chloride. The further metabolism of methyltetrahydro-folate to acetate proceeds via the reactions of the acetyl CoA pathway.155,156

Dehalobacterium formicoaceticum, which utilizes dichloromethane assole energy source, ferments DCM to acetate and formate in a molar ratio of1:2.143 Cell extracts in the presence of tetrahydrofolate, ATP, methyl viologen,and H2 were found to convert DCM to methylene tetrahydrofolate. DCM isassumed to react with a reduced Co(I) corrinoid, forming chloride and chloro-methyl-Co(III) corrinoid, which acted as a donor for a methyltransferase, gen-erating chloromethyltetrahydrofolate. The latter spontaneously rearranged toyield chloride and N 5,N 10-methylenetetrahydrofolate.157

5.5. Reductive Dehalogenation

Reductive dehalogenation is a two-electron-transfer reaction that involvesthe release of the halogen as a halogenide ion and its replacement by hy-drogen. The mechanisms of reductive dehalogenation of haloaliphatic com-pounds is not fully understood, although there are a number of reports onthe metabolism of halogenated aliphatic hydrocarbons under methanogenic,sulfate-reducing, and denitrifying conditions.158−171 For the strictly anaerobicmethanogens, Fathepure and Boyd172 presented a scheme linking reductivedechlorination to methanogenisis. In this scheme they proposed that thechlorinated ethylenes serve as electron acceptors. Clostridium strain TCAIIBisolated from a methanogenic mixed culture was found to reductively dechlo-rinate 1,1,1-trichloroethane to 1,1-dichloroethane and dechlorination of tetra-chloromethane to tri- and dichloromethane.173 There is evidence for reduc-tive dehalogenation under methanogenic, sulfidogenic, and even denitrifyingconditions of a number of haloaromatics such as chlorobenzenes, chloro-toluenes, chlorobenzoates, 2,4 dichlorobenzoate, a number of chlorinatedphenols, tri- and tetrachlorocatechols, di-, tri-, and tetrachloroanilines, 2,4,5-trichlorophenoxyacetic, acid and polychlorinated biphenyls. In the reductivedechlorination mechanism, a reduced organic substrate or H2 might be thesource of both the reducing power and the protons.6

Biotransformation of many halogenated pesticides has been known toinvolve reductive dehalogenation. A list of halogenated pesticides (mostof which are chlorinated) and anthropogenic compounds undergoing re-ductive dehalogenation was presented by Kobayashi and Rittmann174 andMohn and Tiedje.175 Desulfomonile tiedjei DCB-1 reductively dechlorinates3-chlorobenzoate, meta-substituted dichlorobenzoates, chlorophenols, andtetrachloroethylene.176−179 Clostridium rectum S-17, C. sphenoides, several

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Biodegradation of Chlorinated Compounds 185

Bacillus strains, and members of the family Enterobacteriaceae are involvedin reductive dechlorination of lindane.180−186

A metabolic pathway of DDT dechlorination by Aerobacter aero-genes involving reductive and dehydrochlorination steps, yielding 4,4′-dichlorobenzophenone, was proposed by Wedemeyer.187 Dicamba, afterdemethylation, was reductively dechlorinated to 6-chlorosalicylate by ananaerobic consortium.188

6. MOLECULAR BIOLOGY INVOLVED IN DEGRADATION

Several genes, plasmids, and transposons that are involved in the biodegra-dation of chlorinated compounds have been characterized. Alcaligenes sp.strain BR60 carries a 3-chlorobenzoate catabolic gene (3Cba) on a 17-kb trans-poson (Tn 5271) found on an 85-kb broad-host-range plasmid pBRC60.189

Brenner et al.124 showed that of the two separate dioxygenases involved indechlorination in Pseudomonas putida p111, one was chromosomally codedwhile the other chlorobenzoate dioxygenase was shown to be coded on aplasmid pPb111 (75 kb).

Differences in congener specificity between bacteria reflect differencesin the bhp genes. The bhp genes encode PCB encoding enzymes in sev-eral organisms like Pseudomonas sp.190 Expression of a chlorocatechol 1,2-dioxygenase and chlorocatechcol 2,3-dioxygenase genes in chlorobenzene-contaminated subsurface samples was studied by Alfreider et al.191 In astimulation experiment carried out by the authors, hydrogen peroxide wasadded as an oxygen-releasing compound; RNA was directly extracted andused for reverse-transcription polymerase chain reaction (PCR) to analyzethe expression of genes encoding enzymes involved in the catabolism ofchlorobenzene. A study by Springael et al.192 demonstrated the occurrence ofa Tn 4371-related mobile element and sequence in chlorobiphenyl degradingbacteria. Tn 4371 is a 55-kb transposable element involved in the degrada-tion of biphenyl or 4-chlorobiphenyl identified in Ralstonia eutrophea A5. Itdisplays a modular structure including a phage-like integrase gene (int), aPseudomonas-like (chloro) biphenyl catabolic gene cluster (bhp), and RP4-and Ti plasmid-like transfer genes (trb).192 Cloning and characterization oflin genes responsible for the degradation of hexachorocyclohexane isomersby Shpingomonas paucimobilis strain B90 was carried out by Rekha et al.193

Two nonidentical copies of the lin A gene encoding HCH dehydrochlorinase,which were designated linA1 and linA2, were found in this bacteria. Cloningand characterization of lin B, lin C, and lin X were also carried out.193 To im-prove the capabilities of microorganisms relevant for biodegradation, a newgenetic approach was developed by Yoshiyuki et al.,194 who applied it to

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the bhp operon of Pseudomonas sp. strain KKS102 to enhance its biphenyl(PCB)-degrading activity.

7. CONCLUSION AND FUTURE RESEARCH NEEDS

The fate of synthetic chemicals reaching the environment for the most partdepends on the microorganisms present in that part of the environment. Thecapacity of microbes to produce enzymes that recognize xenobiotic com-pounds and to catalyze reactions that break them decides the extent to whichsuch chemicals can cause damage to the ecosystem. The absence of microor-ganisms or microbial systems that bring about their degradation will only re-sult in these chemicals being recalcitrant, persistent, and a potent hazard tothe ecosystem as a whole. Microbial degradation of chlorinated compoundsin general can be divided into cometabolic conversions or conversions thatyield energy and are metabolically productive. Chlorinated aliphatics over arange act as the sole source of carbon and energy to different groups of bac-teria. Since chlorinated compounds are electron deficient, they act as electronacceptors and energy is generated in a respiratory process in anaerobic envi-ronments. In fortuitous metabolism, the chlorinated compound is degradedonly in the presence of another substrate and is degraded due to the pres-ence of broad-specificity enzymes present in bacteria. Enzymes produced byorganisms that degrade chlorinated compounds are coded by genes that ei-ther are chromosomally carried or are present on plasmids. Many of them aretransposable elements. A vast number of such genes have been characterizedand strategies for engineered organisms that carry genes for biodegradationhave been constructed.

Future research related to biodegradation of chlorinated compoundsshould focus on both basic and applied aspects of the subject. Since biore-mediation is an important tool in detoxifying and eliminating environmentalcontaminants, a thorough understanding of microbial genetics, biochemistry,and physiology is required. Attempts should be made to bridge the gap be-tween success at laboratory level and success of the same at a field scale.Many times, laboratory testing doesn’t accurately predict field results for manyprocesses. The reason for the most part is attributed to differences in physi-ological conditions, concentration of the target chemical, and other physical,chemical, and microbial aspects that either were not taken into considera-tion or show constant variation. Research should focus on studies that arecloser to “real” field or ground conditions. The concentration of the tar-get chemicals used for carrying out biodegradation studies in the laboratoryshould not be hypothetical but should relate to contamination levels presentin the environment. Further, treatment of hazardous chemicals in the envi-ronment also presents the possibility of unknown by-products of biodegra-dation entering the environment. Consequently, sound knowledge of the

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Biodegradation of Chlorinated Compounds 187

degradation products, metabolic pathway, biochemistry, and other detailsrelating to treatability studies should be collected before venturing into afull-scale bioremediation process. Most of the research reported on degra-dation of chlorinated compounds is limited to flask experiments, and thereis a need to also develop suitable bioreactor systems for treatment of wastecontaining high concentrations of chlorinated compounds emitted from man-ufacturing industries. A lot has been done and a lot needs to be done in thisdirection.

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