Biotransformation of 1,1,1-trichloroethane, trichloromethane, and tetrachloromethane by a Clostridium sp

A gram-positive, strictly anaerobic, motile, endospore-forming rod, tentatively identified as a proteolytic Clostridium sp., was isolated from the effluent of an anaerobic suspended-growth bioreactor. The organism was able to biotransform 1,1,1-trichloroethane, trichloromethane, and tetrachloromethane. 1,1,1-Trichloroethane was completely transformed (greater than or equal to 99.5%) by reductive dehalogenation to 1,1-dichloroethane (30 to 40%) and, presumably by other mechanisms, to acetic acid (7%) and unidentified products. The reductive dehalogenation of tetrachloromethane led to the intermediate trichloromethane, which was further transformed to dichloromethane (8%) and unidentified products. The biotransformation occurred during the exponential growth phase, as well as during the stationary phase. Tetrachlorethene, trichloroethene, 1,1-dichloroethene, chloroethane, 1,1-dichloroethane, and dichloromethane were not biotransformed significantly by the organism.     

Transformation of tetrachloromethane to dichloromethane and carbon dioxide by Acetobacterium woodii.

Five anaerobic bacteria were tested for their abilities to transform tetrachloromethane so that information about enzymes involved in reductive dehalogenations of polychloromethanes could be obtained. Cultures of the sulfate reducer Desulfobacterium autotrophicum transformed some 80 microM tetrachloromethane to trichloromethane and a small amount of dichloromethane in 18 days under conditions of heterotrophic growth. The acetogens Acetobacterium woodii and Clostridium thermoaceticum in fructose-salts and glucose-salts media, respectively, degraded some 80 microM tetrachloromethane completely within 3 days. Trichloromethane accumulated as a transient intermediate, but the only chlorinated methanes recovered at the end of the incubation were 8 microM dichloromethane and traces of chloromethane. Desulfobacter hydrogenophilus and an autotrophic, nitrate-reducing bacterium were unable to transform tetrachloromethane. Reduction of chlorinated methanes was thus observed only in the organisms with the acetyl-coenzyme A pathway. Experiments with [14C]tetrachloromethane were done to determine the fate of this compound in the acetogen A. woodii. Radioactivity in an 11-day heterotrophic culture was largely (67%) recovered in CO2, acetate, pyruvate, and cell material. In experiments with cell suspensions to which [14C]tetrachloromethane was added, 14CO2 appeared within 20 s as the major transformation product. A. woodii thus catalyzes reductive dechlorinations and transforms tetrachloromethane to CO2 by a series of unknown reactions.     

Transformation of tetrachloromethane to dichloromethane and carbon dioxide by Acetobacterium woodii

Five anaerobic bacteria were tested for their abilities to transform tetrachloromethane so that information about enzymes involved in reductive dehalogenations of polychloromethanes could be obtained. Cultures of the sulfate reducer Desulfobacterium autotrophicum transformed some 80 microM tetrachloromethane to trichloromethane and a small amount of dichloromethane in 18 days under conditions of heterotrophic growth. The acetogens Acetobacterium woodii and Clostridium thermoaceticum in fructose-salts and glucose-salts media, respectively, degraded some 80 microM tetrachloromethane completely within 3 days. Trichloromethane accumulated as a transient intermediate, but the only chlorinated methanes recovered at the end of the incubation were 8 microM dichloromethane and traces of chloromethane. Desulfobacter hydrogenophilus and an autotrophic, nitrate-reducing bacterium were unable to transform tetrachloromethane. Reduction of chlorinated methanes was thus observed only in the organisms with the acetyl-coenzyme A pathway. Experiments with [14C]tetrachloromethane were done to determine the fate of this compound in the acetogen A. woodii. Radioactivity in an 11-day heterotrophic culture was largely (67%) recovered in CO2, acetate, pyruvate, and cell material. In experiments with cell suspensions to which [14C]tetrachloromethane was added, 14CO2 appeared within 20 s as the major transformation product. A. woodii thus catalyzes reductive dechlorinations and transforms tetrachloromethane to CO2 by a series of unknown reactions.     

Anaerobic transformation of 1,1,1-trichloroethane by municipal digester sludge

Anaerobic transformations of 1,1,1-trichloroethane (TCA), 1,1-dichloroethane (DCA), and chloroethane (CA) were studied with sludge from a lab-scale, municipal wastewater sludge digester. TCA was biologically transformed to DCA and CA and further to ethane by reductive dechlorination. TCA was also converted to acetic acid and 1,1-dichloroethene (11DCE) by cell-free extract. 11DCE was further biologically converted to ethene. This pathway was confirmed by transformation tests of TCA, DCA and CA, by tests with cell-free extract, and by chloride release during TCA degradation. With cell-free extract, acetic acid accounted for approximately 90% of the TCA transformed; tests with live cells indicate that the fraction of TCA transformed by this pathway decreased with lower biomass. The dechlorination of DCA to CA and CA to ethane was not stoichiometric. A high rate of TCA removal was observed under the experimental conditions. The results indicate that removal of TCA in anaerobic digestion should be complete, but DCA and CA could persist in a normally operating digester.     

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B₁ and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.     

Anaerobic dechlorination of trichloroethene,tetrachloroethene and 1,2-dichloroethane by an acetogenic mixed culture in a fixed-bed reactor

An anaerobic enrichment culture with glucose as the sole source of carbon and energy plus trichloroethene (TCE) as a potential electron acceptor was inoculated with material from a full size anaerobic charcoal reactor that biologically eliminated dichloromethane from contaminated groundwater (Stromeyer et al. 1991). In subcultures of this enrichment complete sequential transformation of 10 µM TCE viacis-dichloroethene and chloroethene to ethene was reproducibly observed. Maintenance of this activity on subcultivation required the presence of TCE in the medium. The enrichment culture was used to inoculate an anaerobic fixed-bed reactor containing sintered glass Raschig elements as support material. The reactor had a total volume of 1780 ml and was operated at 20 °C in an up-flow mode with a flow rate of 50 ml/h. It was fed continuously with 2 mM glucose and 55 µM TCE. Glucose was converted to acetate as the major product and to a minor amount of methane; TCE was quantitatively dehalogenated to ethene. When, in addition to TCE, tetrachloroethene or 1,2-dichloroethane were added to the system, these compounds were also dehalogenated to ethene. In contrast, 1,1,1-trichloroethane was not dehalogenated, but at 40 µM severely inhibited acetogenesis and methanogenesis. When the concentration of TCE in the feed was raised to 220 µM, chloroethene transiently accumulated, but after an adaptation period ethene was again the only volatile product detected in the effluent. The volumetric degradation rate at this stage amounted to 6.2 µmol/l/h. Since complete transformation of TCE occurred in the first sixth of the reactor volume, the degradation capacity of the system is estimated to exceed this value by factor of about ten.Abbreviations CA chloroethane - 1,1-DCA 1,1-dichloroethane - 1,2-DCA 1,2-dichloroethane - 1,1-DCE 1,1-dichloroethene - c-DCE cis-1,2-dichloroethene - t-DCE trans-1,2-dichloroethene - PCE tetrachloroethene, perchloroethene - 1,1,1-TCA 1,1,1-trichloroethane - TCE trichloroethene - VC chloroethene, vinyl chloride     

Complete transformation of 1,1,1-trichloroethane to chloroethane by a methanogenic mixed population

A methanogenic mixed population in a packed-bed reactor completely transformed 1,1,1-trichloroethane (10 μM) to chloroethane by a cometabolic process. Chloroethane was not further transformed. Acetate and methanol served as electron donors. Complete transformation of 1,1,1-trichloroethane to chloroethane only occurred when sufficient electron donor was fed into the reactor. Otherwise, besides chloroethane, 1,1-dichloroethane was also found as a product. The products of 1,1,1-trichloroethane transformation also depended on the type of electron donor present. With acetate, the degree of dechlorination was higher, i.e. more 1,1,1-trichloroethane was transformed to chloroethane than with methanol. In an enrichment culture obtained from the reactor contents, 1,1,1-trichloroethane was only transformed to 1,1-dichloroethane and was not further metabolized. Methanol, acetate, formate, ethanol, 2-propanol, trimethylamine and H₂, but not dimethylamine and methylamine, served as electron donors for 1,1,1-trichloroethane transformation by this enrichment culture. Both nitrate and nitrite inhibited 1,1,1-trichloroethane transformation; while nitrate completely inhibited 1,1,1-trichloroethane dechlorination, some conversion did occur in the presence of nitrite. The product(s) of this conversion remain unknown, since no chlorinated hydrocarbons were detected. Received: 19 June 1998 / Received revision: 14 September 1998 / Accepted: 17 September 1998     

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.     

Isolation and characterization of Dehalobacterium formicoaceticum gen. nov. sp. nov., a strictly anaerobic bacterium utilizing dichloromethane as source of carbon and energy

A strictly anaerobic, dichloromethane-utilizing bacterium was isolated from a previously described dichloromethane-fermenting, two-component mixed culture. In a mineral medium with vitamins, the organism converted 5 mM dichloromethane within 7 days to formate plus acetate in a molar ratio of 2:1 and to biomass and traces of pyruvate. Of 50 potential substrates and combinations of substrates tested, only dichloromethane supported growth. The organism had a DNA G+C content of 42.7 mol%. From its phylogenetic position deduced from 16S rDNA analysis and from its unique substrate range, we conclude that the organism represents a new genus and a new species within the phylum of the gram-positive bacteria for which we propose the name Dehalobacterium formicoaceticum. Cell extracts were found to contain carbon monoxide dehydrogenase, methylene tetrahydrofolate dehydrogenase, formyl tetrahydrofolate synthetase, and hydrogenase activities, whereas activities of methenyl tetrahydrofolate cyclohydrolase and methylene tetrahydrofolate reductase were not detectable. Activity for dehalogenation of dichloromethane was lost on preparation of cell extracts, but was maintained in cell suspensions. Oxygen and reagents that react with thiol groups caused irreversible inhibition, and propyl iodide caused reversible inhibition of dehalogenation. Our observations suggest: 1) conversion of dichloromethane to methylene tetrahydrofolate, which gives rise to both formate and the methyl group of acetate, or 2) conversion of two molecules of dichloromethane to methylene tetrahydrofolate (which is oxidized to formate) and parallel reductive dehalogenation of one dichloromethane to the methyl group of the corrinoid-protein involved in acetate formation. Received: 11 March 1996 / Accepted 3 May 1996     

A 1,1,1-trichloroethane-degrading anaerobic mixed microbial culture enhances biotransformation of mixtures of chlorinated ethenes and ethanes

1,1,1-trichloroethane (1,1,1-TCA) is a common groundwater pollutant as a result of improper disposal and accidental spills. It is often found as a cocontaminant with trichloroethene (TCE) and inhibits some TCE-degrading microorganisms. 1,1,1-TCA removal is therefore required for effective bioremediation of sites contaminated with mixed chlorinated organics. This study characterized MS, a 1,1,1-TCA-degrading, anaerobic, mixed microbial culture derived from a 1,1,1-TCA-contaminated site in the northeastern United States. MS reductively dechlorinated 1,1,1-TCA to 1,1-dichloroethane (1,1-DCA) and then to monochloroethane (CA) but not further. Cloning of bacterial 16S rRNA genes revealed among other organisms the presence of a Dehalobacter sp. and a Desulfovibrio sp., which are both phylogenetically related to known dehalorespiring strains. Monitoring of these populations with species-specific quantitative PCR during degradation of 1,1,1-TCA and 1,1-DCA showed that Dehalobacter proliferated during dechlorination. Dehalobacter growth was dechlorination dependent, whereas Desulfovibrio growth was dechlorination independent. Experiments were also performed to test whether MS could enhance TCE degradation in the presence of inhibiting levels of 1,1,1-TCA. Dechlorination of cis-dichloroethene (cDCE) and vinyl chloride (VC) in KB-1, a chloroethene-degrading culture used for bioaugmentation, was inhibited with 1,1,1-TCA present. When KB-1 and MS were coinoculated, degradation of cDCE and VC to ethene proceeded as soon as the 1,1,1-TCA was dechlorinated to 1,1-DCA by MS. This demonstrated the potential application of the MS and KB-1 cultures for cobioaugmentation of sites cocontaminated with 1,1,1-TCA and TCE.     

A 1,1,1-Trichloroethane-Degrading Anaerobic Mixed Microbial Culture Enhances Biotransformation of Mixtures of Chlorinated Ethenes and Ethanes

1,1,1-Trichloroethane (1,1,1-TCA) is a common groundwater pollutant as a result of improper disposal and accidental spills. It is often found as a cocontaminant with trichloroethene (TCE) and inhibits some TCE-degrading microorganisms. 1,1,1-TCA removal is therefore required for effective bioremediation of sites contaminated with mixed chlorinated organics. This study characterized MS, a 1,1,1-TCA-degrading, anaerobic, mixed microbial culture derived from a 1,1,1-TCA-contaminated site in the northeastern United States. MS reductively dechlorinated 1,1,1-TCA to 1,1-dichloroethane (1,1-DCA) and then to monochloroethane (CA) but not further. Cloning of bacterial 16S rRNA genes revealed among other organisms the presence of a Dehalobacter sp. and a Desulfovibrio sp., which are both phylogenetically related to known dehalorespiring strains. Monitoring of these populations with species-specific quantitative PCR during degradation of 1,1,1-TCA and 1,1-DCA showed that Dehalobacter proliferated during dechlorination. Dehalobacter growth was dechlorination dependent, whereas Desulfovibrio growth was dechlorination independent. Experiments were also performed to test whether MS could enhance TCE degradation in the presence of inhibiting levels of 1,1,1-TCA. Dechlorination of cis-dichloroethene (cDCE) and vinyl chloride (VC) in KB-1, a chloroethene-degrading culture used for bioaugmentation, was inhibited with 1,1,1-TCA present. When KB-1 and MS were coinoculated, degradation of cDCE and VC to ethene proceeded as soon as the 1,1,1-TCA was dechlorinated to 1,1-DCA by MS. This demonstrated the potential application of the MS and KB-1 cultures for cobioaugmentation of sites cocontaminated with 1,1,1-TCA and TCE.     

Metabolism of chlorofluorocarbons and polybrominated compounds by Pseudomonas putida G786(pHG-2) via an engineered metabolic pathway.

The recombinant bacterium Pseudomonas putida G786(pHG-2) metabolizes pentachloroethane to glyoxylate and carbon dioxide, using cytochrome P-450CAM and toluene dioxygenase to catalyze consecutive reductive and oxidative dehalogenation reactions (L.P. Wackett, M.J. Sadowsky, L.N. Newman, H.-G. Hur, and S. Li, Nature [London] 368:627-629, 1994). The present study investigated metabolism of brominated and chlorofluorocarbon compounds by the recombinant strain. Under anaerobic conditions, P. putida G786(pHG-2) reduced 1,1,2,2-tetrabromoethane, 1,2-dibromo-1,2-dichloroethane, and 1,1,1,2-tetrachloro-2,2-difluoroethane to products bearing fewer halogen substituents. Under aerobic conditions, P. putida G786(pHG-2) oxidized cis- and trans-1,2-dibromoethenes, 1,1-dichloro-2,2-difluoroethene, and 1,2-dichloro-1-fluoroethene. Several compounds were metabolized by sequential reductive and oxidative reactions via the constructed metabolic pathway. For example, 1,1,2,2-tetrabromoethane was reduced by cytochrome P-450CAM to 1,2-dibromoethenes, which were subsequently oxidized by toluene dioxygenase. The same pathway metabolized 1,1,1,2-tetrachloro-2,2-difluoroethane to oxalic acid as one of the final products. The results obtained in this study indicate that P. putida G786(pHG-2) metabolizes polyfluorinated, chlorinated, and brominated compounds and further demonstrates the value of using a knowledge of catabolic enzymes and recombinant DNA technology to construct useful metabolic pathways.     

Substrate interactions in dehalogenation of 1,2-dichloroethane, 1,2-dichloropropane,and 1,1,2-trichloroethane mixtures by Dehalogenimonas spp.

When chlorinated alkanes are present as soil or groundwater pollutants, they often occur in mixtures. This study evaluated substrate interactions during the anaerobic reductive dehalogenation of chlorinated alkanes by the type strains of two Dehalogenimonas species, D. lykanthroporepellens and D. alkenigignens. Four contaminant mixtures comprised of combinations of the chlorinated solvents 1,2-dichloroethane (1,2-DCA), 1,2-dichloropropane (1,2-DCP), and 1,1,2-trichloroethane (1,1,2-TCA) were assessed for each species. Chlorinated solvent depletion and daughter product formation determined as a function of time following inoculation into anaerobic media revealed preferential dechlorination of 1,1,2-TCA over both 1,2-DCA and 1,2-DCP for both species. 1,2-DCA in particular was not dechlorinated until 1,1,2-TCA reached low concentrations. In contrast, both species concurrently dechlorinated 1,2-DCA and 1,2-DCP over a comparably large concentration range. This is the first report of substrate interactions during chlorinated alkane dehalogenation by pure cultures, and the results provide insights into the chlorinated alkane transformation processes that may be expected for contaminant mixtures in environments where Dehalogenimonas spp. are present.     

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     

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.     

Kinetics of biotransformation of 1,1,1-trichloroethane by Clostridium sp. strain TCAIIB

Batch experiments were conducted to examine the effects of high concentrations of 1,1,1-trichloroethane (TCA) on the biotransformation of TCA by Clostridium sp. strain TCAIIB. The biotic dehalogenation of TCA to 1,1-dichloroethane by nongrowing cells was measured at 35 degrees C, and the data were used to obtain the kinetic parameters of the Monod relationship half-velocity coefficient Ks (31 microM) and the coefficient of maximum rate of TCA biotransformation (kTCA; 0.28 mumol per mg per day). The yield of biomass decreased with an increase in the TCA concentration, although TCA concentrations up to 750 microM did not completely inhibit bacterial growth. Also, kTCA was higher in the presence of high concentrations of TCA. A mathematical model based on a modified Monod equation was used to describe the biotransformation of TCA. The abiotic transformation of TCA to 1,1-dichloroethene was measured at 35 degrees C, and the first-order formation rate coefficient for 1,1-dichloroethene (ke) was determined to be 0.86 per year.     

Kinetics of biotransformation of 1,1,1-trichloroethane by Clostridium sp. strain TCAIIB.

Batch experiments were conducted to examine the effects of high concentrations of 1,1,1-trichloroethane (TCA) on the biotransformation of TCA by Clostridium sp. strain TCAIIB. The biotic dehalogenation of TCA to 1,1-dichloroethane by nongrowing cells was measured at 35 degrees C, and the data were used to obtain the kinetic parameters of the Monod relationship half-velocity coefficient Ks (31 microM) and the coefficient of maximum rate of TCA biotransformation (kTCA; 0.28 mumol per mg per day). The yield of biomass decreased with an increase in the TCA concentration, although TCA concentrations up to 750 microM did not completely inhibit bacterial growth. Also, kTCA was higher in the presence of high concentrations of TCA. A mathematical model based on a modified Monod equation was used to describe the biotransformation of TCA. The abiotic transformation of TCA to 1,1-dichloroethene was measured at 35 degrees C, and the first-order formation rate coefficient for 1,1-dichloroethene (ke) was determined to be 0.86 per year.     

Complete Reductive Dechlorination of 1,2-Dichloropropane by Anaerobic Bacteria

The transformation of 1,2-dichloropropane (1,2-D) was observed in anaerobic microcosms and enrichment cultures derived from Red Cedar Creek sediment. 1-Chloropropane (1-CP) and 2-CP were detected after an incubation period of 4 weeks. After 4 months the initial amount of 1,2-D was stoichiometrically converted to propene, which was not further transformed. Dechlorination of 1,2-D was not inhibited by 2-bromoethanesulfonate. Sequential 5% (vol/vol) transfers from active microcosms yielded a sediment-free, nonmethanogenic culture, which completely dechlorinated 1,2-D to propene at a rate of 5 nmol min(sup-1) mg of protein(sup-1). No intermediate formation of 1-CP or 2-CP was detected in the sediment-free enrichment culture. A variety of electron donors, including hydrogen, supported reductive dechlorination of 1,2-D. The highest dechlorination rates were observed between 20(deg) and 25(deg)C. In the presence of 1,2-D, the hydrogen threshold concentration was below 1 ppm by volume (ppmv). In addition to 1,2-D, the enrichment culture transformed 1,1-D, 2-bromo-1-CP, tetrachloroethene, 1,1,2,2-tetrachloroethane, and 1,2-dichloroethane to less halogenated compounds. These findings extend our knowledge of the reductive dechlorination process and show that halogenated propanes can be completely dechlorinated by anaerobic bacteria.     

In Vitro Studies on Reductive Vinyl Chloride Dehalogenation by an Anaerobic Mixed Culture

Reductive dehalogenation of vinyl chloride (VC) was studied in an anaerobic mixed bacterial culture. In growth experiments, ethene formation from VC increased exponentially at a rate of about 0.019 h(sup-1). Reductive VC dehalogenation was measured in vitro by using cell extracts of the mixed culture. The apparent K(infm) for VC was determined to be about 76 (mu)M; the V(infmax) was about 28 nmol (middot) min(sup-1) (middot) mg of protein(sup-1). The VC-dehalogenating activity was membrane associated. Propyl iodide had an inhibitory effect on the VC-dehalogenating activity in the in vitro assay. However, this inhibition could not be reversed by illumination. Cell extracts also catalyzed the reductive dehalogenation of cis-1,2-dichloroethene (cis-DCE) and, at a lower rate, of trichloroethene (TCE). Tetrachloroethene (PCE) was not transformed. The results indicate that the reductive dehalogenation of VC and cis-DCE described here is different from previously reported reductive dehalogenation of PCE and TCE.