Bacterial dehalogenation

Halogenated organic compounds are produced industrially in large quantities and represent an important class of environmental pollutants. However, an abundance of haloorganic compounds is also produced naturally. Bacteria have evolved several strategies for the enzyme-catalyzed dehalogenation and degradation of both haloaliphatic and haloaromatic compounds: (i) Oxidative dehalogenation is the result of mono- or dioxygenase-catalyzed, co-metabolic or metabolic reactions. (ii) In dehydrohalogenase-catalyzed dehalogenation, halide elimination leads to the formation of a double bond. (iii) Substitutive dehalogenation in most cases is a hydrolytic process, catalyzed by halidohydrolases, but there also is a “thiolytic” mechanism with glutathione as cosubstrate. Dehalogenation by halohydrin hydrogen-halide lyases is the result of an intramolecular substitution reaction. (iv) A distinct dechlorination mechanism involves methyl transfer from chloromethane onto tetrahydrofolate. (v) Reductive dehalogenations are co-metabolic processes, or they are specific reactions involved in substrate utilization (carbon metabolism), or reductive dehalogenation is coupled to energy conservation: some anaerobic bacteria use a specific haloorganic compound as electron acceptor of a respiratory process. This review discusses the mechanisms of enzyme-catalyzed dehalogenation reactions, describes some pathways of the bacterial degradation of haloorganic compounds, and indicates some trends in the biological treatment of organohalogen-polluted air, groundwater, soil, and sediments. Received: 24 June 1998 / Received revision: 1 September 1998 / Accepted: 3 September 1998     

Cosubstrate effects in reductive dehalogenation byPseudomonas putida G786 expressing cytochrome P-450CAM

Cytochrome P-450_CAM was shown to be the primary catalyst mediating reductive dehalogenation of polychlorinated ethanes byPseudomonas putida G786. Under anaerobic conditions, the enzyme catalyzed reductive elimination reactionsin vivo with the substrates hexachloroethane, pentachloroethane, and 1,1,1,2-tetrachloroethane; the products were tetrachloroethylene, trichloroethylene, and 1,1-dichloroethylene, respectively.In vivo reaction rates were determined. No reaction was observed with 1,1,2,2-tetrachloroethane or 1,1,1-trichloroethane. Purified cytochrome P-450_CAM was used to measure dissociation constants of polychlorinated ethanes for the enzyme active site. Observed rates and dissociation constants were used to predict the course of a reaction with the three substrates simultaneously. Data obtained from experiments withP. putida G786 generally followed the simulated reaction curves. Oxygen suppressed the reductive dechlorination reactions and, in the case of 1,1,1,2-tetrachloroethane, 2,2,2-trichloroacetaldehyde was formed. Significant rates of reductive dechlorination were observed at 5% oxygen suggesting that these reactions could occur under partially aerobic conditions. These studies highlight the potential to use an aerobic bacterium,P. putida G786, under a range of oxygen tensions to reductively dehalogenate mixed wastes which are only degraded at very low rates by obligately anaerobic bacteria.Abbreviations GC/MS Gas chromatography/mass spectrometry - P-450_CAM Cytochrome m of the camphor oxidizing system ofP. putida - pca Polychlorinated ethane     

Chloroform respiration to dichloromethane by a Dehalobacter population

Chloroform (CF), or trichloromethane, is an ubiquitous environmental pollutant because of its widespread industrial use, historically poor disposal and recalcitrance to biodegradation. Chloroform is a potent inhibitor of metabolism and no known organism uses it as a growth substrate. We discovered that CF was rapidly and sustainably dechlorinated in the course of investigating anaerobic reductive dechlorination of 1,1,1‐trichloroethane in a Dehalobacter‐containing culture. Like 1,1,1‐trichloroethane dechlorination in this culture, CF dechlorination was a growth‐linked respiratory process, requiring H₂ as an electron donor and CF as an electron acceptor. Moreover, the same specific reductive dehalogenase likely catalyzed both reactions. This Dehalobacter population appears specialized for substrates with three halogen substituents on the same carbon atom, with widespread implications for bioremediation.     

Microbial transformation of chlorinated benzoates

Chlorinated benzoates enter the environment through their use as herbicides or as metabolites of other halogenated compounds. Ample evidence is available indicating biodegradation of chlorinated benzoates to CO₂ and chloride in the environment under aerobic as well as anaerobic conditions. Under aerobic conditions, lower chlorinated benzoates can serve as sole electron and carbon sources supporting growth of a large list of taxonomically diverse bacterial strains. These bacteria utilize a variety of pathways ranging from those involving an initial degradative attack by dioxygenases to those initiated by hydrolytic dehalogenases. In addition to monochlorinated benzoates, several bacterial strains have been isolated that can grow on dichloro-, and trichloro- isomers of chlorobenzoates. Some aerobic bacteria are capable of cometabolizing chlorinated benzoates with simple primary substrates such as benzoate. Under anaerobic conditions, chlorinated benzoates are subject to reductive dechlorination when suitable electron-donating substrates are available. Several halorespiring bacteria are known which can use chlorobenzoates as electron acceptors to support growth. For example, Desulfomonile tiedjei catalyzes the reductive dechlorination of 3-chlorobenzoate to benzoate. The benzoate skeleton is mineralized by other microorganisms in the anaerobic environment. Various dichloro- and trichlorobenzoates are also known to be dechlorinated in anaerobic sediments.     

Microbial reductive dehalogenation of polychlorinated biphenyls

Under anaerobic conditions, microbial reductive dechlorination of polychlorinated biphenyls (PCBs) occurs in soils and aquatic sediments. In contrast to dechlorination of supplemented single congeners for which frequently ortho dechlorination has been observed, reductive dechlorination mainly attacks meta and/or para chlorines of PCB mixtures in contaminated sediments, although in a few instances ortho dechlorination of PCBs has been observed. Different microorganisms appear to be responsible for different dechlorination activities and the occurrence of various dehalogenation routes. No axenic cultures of an anaerobic microorganism have been obtained so far. Most probable number determinations indicate that the addition of PCB congeners, as potential electron acceptors, stimulates the growth of PCB-dechlorinating microorganisms. A few PCB-dechlorinating enrichment cultures have been obtained and partially characterized. Temperature, pH, availability of naturally occurring or of supplemented carbon sources, and the presence or absence of H(2) or other electron donors and competing electron acceptors influence the dechlorination rate, extent and route of PCB dechlorination. We conclude from the sum of the experimental data that these factors influence apparently the composition of the active microbial community and thus the routes, the rates and the extent of the dehalogenation. The observed effects are due to the specificity of the dehalogenating bacteria which become active as well as changing interactions between the dehalogenating and non-dehalogenating bacteria. Important interactions include the induced changes in the formation and utilization of H(2) by non-dechlorinating and dechlorinating bacteria, competition for substrates and other electron donors and acceptors, and changes in the formation of acidic fermentation products by heterotrophic and autotrophic acidogenic bacteria leading to changes in the pH of the sediments.     

Complete dechlorination of tetrachlorohydroquinone by cell extracts of pentachlorophenol-induced Rhodococcus chlorophenolicus.

In this paper we describe the sequence of reactions leading from tetrachloro-para-hydroquinone to 1,2,4-trihydroxybenzene by inducible enzymes of Rhodococcus chlorophenolicus. Tetrachlorohydroquinone was first converted to a dichlorotrihydroxybenzene in a reaction involving both hydrolytic and reductive dechlorination; no trichlorinated intermediate was detected. Dichlorotrihydroxybenzene was subsequently reductively dechlorinated to a monochlorotrihydroxybenzene and finally to 1,2,4-trihydroxybenzene. The cell extract also catalyzed, at a lower rate, reductive dechlorination of trichlorohydroquinone, mainly to 2,3-dichlorohydroquinone. To our knowledge this is the first demonstration of reductive aromatic dechlorination by bacterial enzymes.     

Key enzymes in the anaerobic aromatic metabolism catalysing Birch-like reductions

Several novel enzyme reactions have recently been discovered in the aromatic metabolism of anaerobic bacteria. Many of these reactions appear to be catalyzed by oxygen-sensitive enzymes by means of highly reactive radical intermediates. This contribution deals with two key reactions in this metabolism: the ATP-driven reductive dearomatisation of the benzene ring and the reductive removal of a phenolic hydroxyl group. The two reactions catalyzed by benzoyl-CoA reductase (BCR) and 4-hydroxybenzoyl-CoA reductase (4-HBCR) are both mechanistically difficult to achieve; both are considered to proceed in 'Birch-like' reductions involving single electron and proton transfer steps to the aromatic ring. The problem of both reactions is the extremely high redox barrier for the first electron transfer to the substrate (e.g., -1.9 V in case of a benzoyl-CoA (BCoA) analogue), which is solved in the two enzymes in different manners. Studying these enzymatic reactions provides insights into general principles of how oxygen-dependent reactions are replaced by alternative processes under anoxic conditions.     

Transformation of tetra- and trichloromethane to CO2 by anaerobic bacteria is a non-enzymic process

Abstract Degradation of tetrachloromethane was examined in the three strictly anaerobic bacteria, Acetobacterium woodii, Desulfobacterium autotrophicum and Methanobacterium thermoautotrophicum When incubated under anaerobic conditions in reduced buffer, suspensions of each organism degraded CCl₄ by both reductive and substitutive mechanisms. The products formed included less-highly chlorinated methanes and CO₂. Cell-free extracts of A. woodii degraded tetrachloromethane in a manner similar to that in whole cells but at a lower rate (63 vs. 110 μkat/kg of protein). When M. thermoautotrophicum or A. woodii was autoclaved, reductive dechlorination was partly abolished, whereas substitutive dechlorination was retained. Trichloromethane was oxidized to CO₂ by both native and autoclaved cells of A. woodii . Halomethanes are thus degraded anaerobically by reductive, substitutive and oxidative mechanisms.     

Sequential anaerobic/aerobic biodegradation of chloroethenes--aspects of field application

Because of a range of different industrial activities, sites contaminated with chloroethenes are a world-wide problem. Chloroethenes can be biodegraded by reductive dechlorination under anaerobic conditions as well as by oxidation under aerobic conditions. The tendency of chloroethenes to undergo reductive dechlorination decreases with a decreasing number of chlorine substituents, whereas with less chlorine substituents chloroethenes more easily undergo oxidative degradation. There is currently a growing interest in aerobic metabolic degradation of chloroethenes, which demonstrates advantages compared to cometabolic degradation pathways. Sequential anaerobic/aerobic biodegradation can overcome the disadvantages of reductive dechlorination and leads to complete mineralization of the chlorinated pollutants. This approach shows promise for site remediation in natural settings and in engineered systems.     

Key enzymes in the anaerobic aromatic metabolism catalysing Birch-like reductions

Several novel enzyme reactions have recently been discovered in the aromatic metabolism of anaerobic bacteria. Many of these reactions appear to be catalyzed by oxygen-sensitive enzymes by means of highly reactive radical intermediates. This contribution deals with two key reactions in this metabolism: the ATP-driven reductive dearomatisation of the benzene ring and the reductive removal of a phenolic hydroxyl group. The two reactions catalyzed by benzoyl-CoA reductase (BCR) and 4-hydroxybenzoyl-CoA reductase (4-HBCR) are both mechanistically difficult to achieve; both are considered to proceed in ‘Birch-like’ reductions involving single electron and proton transfer steps to the aromatic ring. The problem of both reactions is the extremely high redox barrier for the first electron transfer to the substrate (e.g., −1.9 V in case of a benzoyl-CoA (BCoA) analogue), which is solved in the two enzymes in different manners. Studying these enzymatic reactions provides insights into general principles of how oxygen-dependent reactions are replaced by alternative processes under anoxic conditions.     

Anaerobic Microbial Reductive Dehalogenation of Chlorinated Ethenes

The current knowledge on microbial reductive dechlorination of chlorinated ethenes (CEs) and its application are discussed. Physiological studies on CEs dechlorinating microorganisms indicate that a distinction can be made between cometabolic dechlorination and halorespiration. Whereas cometabolic dechlorination is a coincidental and nonspecific side reaction, catalyzed by several methanogenic and acetogenic bacteria, halorespiration is a specific enzymatic reaction from which metabolic energy can be gained. In contrast to the well-studied biological dechlorination of PCE to cis-DCE, little is known about the biology of the further dechlorination from cis-DCE to ethene. Bacteria performing the latter reaction have not yet been isolated. Microbial reductive dechlorination can be applied to the in situ bioremediation of CEs contaminated sites. From laboratory and field studies, it has become clear that the dechlorination of tetrachloroethene (PCE) to cis-clichloroethene (cis-DCE) occurs rapidly and can be stimulated relatively easily. However, complete reduction to ethene appears to be a slower process that is more difficult to achieve.     

Characterization Studies of an Anaerobic, Pentachlorophenol-Dechlorinating Enrichment Culture

Dechlorination studies were conducted using microbial cultures developed in a fluidized-bed reactor (FBR) that dechlorinates pentachlorophenol (PCP) to 3,4-dichlorophenol (3,4-DCP) and 4-monochlorophenol (4-MCP). Electron donor experiments demonstrated that lactate, propionate, and H₂ can serve as electron donors for chlorophenol (CP) dechlorination in mixed, anaerobic, PCP-enriched cultures. Dechlorination did not proceed in the absence of an electron donor. Acetate, which resulted in little H₂ production, was a poor electron donor. The results of inhibition studies using vancomycin and 2-bromoethanesulfonic acid implicate members of the domain bacteria in the dechlorination of CPs, whereas methanogens do not appear to be involved in dechlorination. Brief heat treatment (80°C for 90 min) of the FBR enrichment cultures implicated endospore formers in the dechlorination of CPs, primarily at the ortho position, where PCP was dechlorinated to 3,4,5-trichlorophenol (3,4,5-TCP) (the sole TCP detected) and subsequently to 3,4-DCP. Both lactate and H₂ served as electron donors in the heat-and oxygen-treated cultures. In contrast, a lactate-fed anaerobic spread-plate enrichment culture exhibited solely meta-dechlorination, where PCP dechlorinated solely to 2,4,6-TCP. The separation of ortho- and meta-specific dechlorination reactions provides evidence that PCP dechlorination in the FBR enrichment culture was catalyzed by at least the following two separate groups of CP-dechlorinating bacteria: one meta-dechlorinating group and one primarily ortho-dechlorinating group.     

Stereoselective microbial dehalorespiration with vicinal dichlorinated alkanes

The suspected carcinogen 1,2-dichloroethane (1,2-DCA) is the most abundant chlorinated C(2) groundwater pollutant on earth. However, a reductive in situ detoxification technology for this compound does not exist. Although anaerobic dehalorespiring bacteria are known to catalyze several dechlorination steps in the reductive-degradation pathway of chlorinated ethenes and ethanes, no appropriate isolates that selectively and metabolically convert them into completely dechlorinated end products in defined growth media have been reported. Here we report on the isolation of Desulfitobacterium dichloroeliminans strain DCA1, a nutritionally defined anaerobic dehalorespiring bacterium that selectively converts 1,2-dichloroethane and all possible vicinal dichloropropanes and -butanes into completely dechlorinated end products. Menaquinone was identified as an essential cofactor for growth of strain DCA1 in pure culture. Strain DCA1 converts chiral chlorosubstrates, revealing the presence of a stereoselective dehalogenase that exclusively catalyzes an energy-conserving anti mechanistic dichloroelimination. Unlike any known dehalorespiring isolate, strain DCA1 does not carry out reductive hydrogenolysis reactions but rather exclusively dichloroeliminates its substrates. This unique dehalorespiratory biochemistry has shown promising application possibilities for bioremediation purposes and fine-chemical synthesis.     

Dependence of tetrachloroethylene dechlorination on methanogenic substrate consumption by Methanosarcina sp. strain DCM.

Tetrachloroethylene (perchloroethylene, PCE) is a suspected carcinogen and a common groundwater contaminant. Although PCE is highly resistant to aerobic biodegradation, it is subject to reductive dechlorination reactions in a variety of anaerobic habitats. The data presented here clearly establish that axenic cultures of Methanosarcina sp. strain DCM dechlorinate PCE to trichloroethylene and that this is a biological reaction. Growth on methanol, acetate, methylamine, and trimethylamine resulted in PCE dechlorination. The reductive dechlorination of PCE occurred only during methanogenesis, and no dechlorination was noted when CH4 production ceased. There was a clear dependence of the extent of PCE dechlorination on the amount of methanogenic substrate (methanol) consumed. The amount of trichloroethylene formed per millimole of CH4 formed remained essentially constant for a 20-fold range of methanol concentrations and for growth on acetate, methylamine, and trimethylamine. These results suggest that the reducing equivalents for PCE dechlorination are derived from CH4 biosynthesis and that the extent of chloroethylene dechlorination can be enhanced by stimulating methanogenesis. It is proposed that electrons transferred during methanogenesis are diverted to PCE by a reduced electron carrier involved in methane formation.     

Constitutive dechlorination of chlorinated ethenes by a methanol degrading methanogenic consortium

The ability of granular methanogenic sludge to dechlorinate chloroethenes was investigated with unadapted sludge from an upflow anaerobic sludge blanket (UASB) reactor fed with methanol. The sludge degraded chlorinated ethenes, but the degradation rates were low. The addition of primary substrate was necessary to sustain dechlorination. The dechlorinating activity seemed to be constitutively present in the anaerobic bacteria. Usually, one chlorine atom was removed via reductive hydrogenolysis. Only trichloroethene (TCE) was converted to substantial amounts of vinylchloride (VC). 1,1-Dichloroethene (1,1DCE) was observed to be an important intermediate in the dechlorination by unadapted granular sludge, although previously this compound had not been commonly observed. Furthermore, the dechlorination of 1,1DCE was faster than the dechlorination of the other chloroethenes.     

Bio-reductive dechlorination of 1,1,1-trichloroethane and chloroform using a hydrogen-based membrane biofilm reactor

A H(2)-based, denitrifying and sulfate-reducing membrane biofilm reactor (MBfR) was effective for removing 1,1,1-trichloroethane (TCA) and chloroform (CF) by reductive dechlorination. When either TCA or CF was first added to the MBfR, reductive dechlorination took place immediately and then increased over 3 weeks, suggesting enrichment for TCA- or CF-dechlorinating bacteria. Increasing the H(2) pressure increased the dechlorination rates of TCA or CF, and it also increased the rate of sulfate reduction. Increased sulfate loading allowed more sulfate reduction, and this competed with reductive dechlorination, particularly the second steps. The acceptor flux normalized by effluent concentration can be an efficient indicator to gauge the intrinsic kinetics of the MBfR biofilms for the different reduction reactions. The analysis of normalized rates showed that the kinetics for reductive-dechlorination reactions were slowed by reduced H(2) bio-availability caused by a low H(2) pressure or competition from sulfate reduction.     

不同电子供体对2,4-二氯酚还原脱氯的影响

以葡萄糖、乙酸钠、Fe0、Fe0 葡萄糖、Fe0 乙酸钠作为电子供体,接种未驯化厌氧混合菌,考察2,4-二氯酚(2,4-DCP)的还原脱氯特性及Fe0作为电子供体的最佳作用条件与持续性特征.结果表明:与葡萄糖的作用相比,Fe0 葡萄糖可有效提高目标物脱氯效果;乙酸钠、Fe0及Fe0 乙酸钠均为有效电子供体,其中Fe0作为电子供体时目标物脱氯效果最佳,最佳作用条件为初始pH8.0,Fe0投加量2.0 g/L,4-CP为其主要脱氯中间产物;Fe0可持续供给2,4-DCP还原脱氯所需电子,而乙酸钠不断消耗后其脱氯效果与Fe0作为电子供体有明显差距.     

Regiospecificity of chlorophenol reductive dechlorination by vitamin b(12s)

Vitamin B(12), reduced by titanium (III) citrate to vitamin B(12s), catalyzes the reductive dechlorination of chlorophenols. Reductive dechlorination of pentachlorophenol and of all tetrachlorophenol and trichlorophenol isomers was observed. Reaction of various chlorophenols with vitamin B(12) favored reductive dechlorination at positions adjacent to another chlorinated carbon, but chlorines ortho to the hydroxyl group of a phenol were particularly resistant to reductive dechlorination, even if they were also ortho to a chlorine. This resulted in a reductive dechlorination pattern favoring removal of para and meta chlorines, which differs substantially from the pattern exhibited by anaerobic microbial consortia.     

Microbial Mineralization of Ethene Under Sulfate-Reducing Conditions

Previous investigations demonstrated that respiratoly reductive dechlorination of vinyl chloride (VC) can be efficient even at H₂ concentrations (≤2 nM) that are characteristic of SO₄-reducing conditions. In the study reported here, microorganisms indigenous to a lake-bed sediment completely mineralized [1,2-¹⁴C] ethene to ¹⁴14CO₂ when incubated under SO₄-reducing conditions. Together, these observations argue for a novel mechanism for the net anaerobic oxidation of VC to CO₂: reductive dechlorination of VC to ethene followed by anaerobic oxidation of ethene to CO₂. Moreover, the results of this study suggest that reliance on ethene and/or ethane accumulation as a quantitative indicator of complete reductive dechlorination of chioroethene contaminants may not be warranted.     

Dearomatizing benzene ring reductases

The high resonance energy of the benzene ring is responsible for the relative resistance of aromatic compounds to biodegradation. Nevertheless, bacteria from nearly all physiological groups have been isolated which utilize aromatic growth substrates as the sole source of cell carbon and energy. The enzymatic dearomatization of the benzene nucleus by microorganisms is accomplished in two different manners. In aerobic bacteria the aromatic ring is dearomatized by oxidation, catalyzed by oxygenases. In contrast, anaerobic bacteria attack the aromatic ring by reductive steps. Key intermediates in the anaerobic aromatic metabolism are benzoyl-CoA and compounds with at least two meta-positioned hydroxyl groups (resorcinol, phloroglucinol and hydroxyhydroquinone). In facultative anaerobes, the reductive dearomatization of the key intermediate benzoyl-CoA requires a stoichiometric coupling to ATP hydrolysis, whereas reduction of the other intermediates is readily achieved with suitable electron donors. Obligately anaerobic bacteria appear to use a totally different enzymology for the reductive dearomatization of benzoyl-CoA including selenocysteine- and molybdenum- containing enzymes.