Dehalococcoides ethenogenes strain 195 dechlorinates tetrachloroethene to vinyl chloride and ethene, and its genome has been found to contain up to 17 putative dehalogenase gene homologues, suggesting diverse dehalogenation ability. We amended pure or mixed cultures containing D. ethenogenes strain 195 with 1,2,3,4-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3-dichlorodibenzo-p-dioxin, 1,2,3,4-tetrachlorodibenzofuran, 2,3,4,5,6-pentachlorobiphenyl, 1,2,3,4-tetrachloronaphthalene, various chlorobenzenes, or a mixture of 2-, 3-, and 4-chlorophenol to determine the dehalogenation ability. D. ethenogenes strain 195 dechlorinated 1,2,3,4-tetrachlorodibenzo-p-dioxin to a mixture of 1,2,4-trichlorodibenzo-p-dioxin and 1,3-dichlorodibenzo-p-dioxin. 2,3,4,5,6- Pentachlorobiphenyl was dechlorinated to 2,3,4,6- and/or 2,3,5,6-tetrachlorobiphenyl and 2,4,6-trichlorobiphenyl. 1,2,3,4-Tetrachloronaphthalene was dechlorinated primarily to an unidentified dichloronaphthalene congener. The predominant end products from hexachlorobenzene dechlorination were 1,2,3,5-tetrachlorobenzene and 1,3,5-trichlorobenzene. We did not detect dechlorination daughter products from monochlorophenols, 2,3-dichlorodibenzo-p-dioxin or 2,3,7,8- tetrachlorodibenzo-p-dioxin. D. ethenogenes strain 195 has the ability to dechlorinate many different types of chlorinated aromatic compounds, in addition to its known chloroethene respiratory electron acceptors. Remediation of sediments contaminated with aromatic halogenated organic pollutants such as polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins could require billions of dollars in the coming years. Harnessing microorganisms such as Dehalococcoides spp. that detoxify these compounds via removal of halogens may lead to cost-effective biotechnological approaches for remediation.

cis-Dichloroethene (DCE) and vinyl chloride (VC) often accumulate in contaminated aquifers in which tetrachloroethene (PCE) or trichloroethene (TCE) undergo reductive dechlorination. "Dehalococcoides ethenogenes" strain 195 is the first isolate capable of dechlorinating chloroethenes past cis-DCE. Strain 195 could utilize commercially synthesized cis-DCE as an electron acceptor, but doses greater than 0.2 mmol/L were inhibitory, especially to PCE utilization. To test whether the cis-DCE itself was toxic, or whether the toxicity was due to impurities in the commercial preparation (97% nominal purity), we produced cis-DCE biologically from PCE using a Desulfitobacterium sp. culture. The biogenic cis-DCE was readily utilized at high concentrations by strain 195 indicating that cis-DCE was not intrinsically inhibitory. Analysis of the commercially synthesized cis-DCE by GC/mass spectrometry indicated the presence of approximately 0.4% mol/mol chloroform. Chloroform was found to be inhibitory to chloroethene utilization by strain 195 and at least partially accounts for the inhibitory activity of the synthetic cis-DCE. VC, a human carcinogen that accumulates to a large extent in cultures of strain 195, was not utilized as a growth substrate, and cultures inoculated into medium with VC required a growth substrate, such as PCE, for substantial VC dechlorination. However, high concentrations of PCE or TCE inhibited VC dechlorination. Use of a hexadecane phase to keep the aqueous PCE concentration low in cultures allowed simultaneous utilization of PCE and VC. At contaminated sites in which "D. ethenogenes" or similar organisms are present, biogenic cis-DCE should be readily dechlorinated, chloroform as a co-contaminant may be inhibitory, and concentrations of PCE and TCE, except perhaps those near the source zone, should allow substantial VC dechlorination.

Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B 12 ) was studied. Fractionation was largest during the reaction with cyanocobalamin with ␣C ‫؍‬ 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (␣C ‫؍‬ 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (␣C ‫؍‬ 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, ␣C values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.The chlorinated ethenes tetrachloroethene (PCE) and trichloroethene (TCE) have been used as solvents in the drycleaning industry and as metal degreasing agents and are among the most common groundwater contaminants due to spillage and leakage (12). The assessment of in situ biodegradation of groundwater contaminants is difficult since a decrease in the concentration can be a result of many factors, including dilution, sorption, and biological conversion. Monitoring and assessment of in situ microbial degradation activities of contaminants at polluted sites is therefore a major challenge. In the last few years, the application of stable isotope techniques has been suggested for assessment of the in situ biodegradation of contaminants (for a review, see references 17 and 33).Stable isotope fractionation of PCE and TCE has been observed under field conditions in contaminated aquifers as well as in laboratory studies by mixed microbial cultures and microcosms (2, 9, 39, 40); however, the factors affecting the isotope fractionation have not yet been studied systematically.Several factors may influence the degree of stable isotope fractionation, including biodegrading microorganisms, properties of the dehalogenating enzymes, and the reaction mechanism. Cobalamins are important cofactors of reductive dehalogenases in organisms capable of dehalorespiration (8,10,15,20,25,26). The microbial dehalogenation by cobalamin-containing dehalogenases has been proposed to proceed by alkylating a superreduced corrinoid containing a Co(I) species at the reactive site with the chloroethene (26). The chemical mechanism of the reductive dehalogenation of chlorinated ethenes catalyzed by cobalamin has been the subject of previous studies and has been suggested to occur via a single elect...

The role of the corrinoid cofactor in reductive dehalogenation catalysis by tetrachloroethene reductive dehalogenase (PceA) of Sulfurospirillum multivorans was investigated using isotope analysis of carbon and chlorine. Crude extracts containing PceA--harboring either a native norpseudo-B12 or the alternative nor-B12 cofactor--were applied for dehalogenation of tetrachloroethene (PCE) or trichloroethene (TCE), and compared to abiotic dehalogenation with the respective purified corrinoids (norpseudovitamin B12 and norvitamin B12), as well as several commercially available cobalamins and cobinamide. Dehalogenation of TCE resulted in a similar extent of C and Cl isotope fractionation, and in similar dual-element isotope slopes (εC/εCl) of 5.0-5.3 for PceA enzyme and 3.7-4.5 for the corrinoids. Both observations support an identical reaction mechanism. For PCE, in contrast, observed C and Cl isotope fractionation was smaller in enzymatic dehalogenation, and dual-element isotope slopes (2.2-2.8) were distinctly different compared to dehalogenation mediated by corrinoids (4.6-7.0). Remarkably, εC/εCl of PCE depended in addition on the corrinoid type: εC/εCl values of 4.6 and 5.0 for vitamin B12 and norvitamin B12 were significantly different compared to values of 6.9 and 7.0 for norpseudovitamin B12 and dicyanocobinamide. Our results therefore suggest mechanistic and/or kinetic differences in catalytic PCE dehalogenation by enzymes and different corrinoids, whereas such differences were not observed for TCE.