Reductive dechlorination of high concentrations of tetrachloroethene to ethene by an anaerobic enrichment culture in the absence of methanogenesis.

Tetrachloroethene, also known as perchloroethylene (PCE), is a common groundwater contaminant throughout the United States. The incomplete reductive dechlorination of PCE--resulting in accumulations of trichloroethene, dichloroethene isomers, and/or vinyl chloride--has been observed by many investigators in a wide variety of methanogenic environments. Previous mixed-culture studies have demonstrated that complete dechlorination to ethene is possible, although the final dechlorination step from vinyl chloride to ethene is rate limiting, with significant levels of vinyl chloride typically persisting. In this study, anaerobic methanol-PCE enrichment cultures which proved capable of dechlorinating high concentrations PCE to ethene were developed. Added concentrations of PCE as high as 550 microM (91-mg/liter nominal concentration; approximately 55-mg/liter actual aqueous concentration) were routinely dechlorinated to 80% ethene and 20% vinyl chloride within 2 days at 35 degrees C. The methanol level used was approximately twice that needed for complete dechlorination of PCE to ethene. The observed transformations occurred in the absence of methanogenesis, which was apparently inhibited by the high concentrations of PCE. When incubation was allowed to proceed for as long as 4 days, virtually complete conversion of PCE to ethene resulted, with less than 1% persisting as vinyl chloride. An electron balance demonstrated that methanol consumption was completely accounted for by dechlorination (31%) and acetate production (69%). The high volumetric rates of PCE dechlorination (up to 275 mumol/liter/day) and the relatively large fraction (ca. one-third) of the supplied electron donor used for dechlorination suggest that reductive dechlorination could be exploited for bioremediation of PCE-contaminated sites.     

Characterization of an H2-utilizing enrichment culture that reductively dechlorinates tetrachloroethene to vinyl chloride and ethene in the absence of methanogenesis and acetogenesis.

We have been studying an anaerobic enrichment culture which, by using methanol as an electron donor, dechlorinates tetrachloroethene (PCE) to vinyl chloride and ethene. Our previous results indicated that H2 was the direct electron donor for rductive dechlorination of PCE by the methanol-PCE culture. Most-probable-number counts performed on this culture indicated low numbers (< or equal to 10(4)/ml)) of methanogens and PCE dechlorinators using methanol and high numbers (> or equal to 10(6)/ml)) of sulfidogens, methanol-utilizing acetogens, fermentative heterotrophs, and PCE dechlorinators using H2. An anaerobic H2-PCE enrichment culture was derived from a 10(-6) dilution of the methanol-PCE culture. This H2-PCE culture used PCE at increasing rates over time when transferred to fresh medium and could be transferred indefinitely with H2 as the electron donor for the PCE dechlorination, indicating that H2-PCE can serve as an electron donor-acceptor pair for energy conservation and growth. Sustained PCE dechlorination by this culture was supported by supplementation with 0.05 mg of vitamin B12 per liter, 25% (vol/vol) anaerobic digestor sludge supernatant, and 2 mM acetate, which presumably served as a carbon source. Neither methanol nor acetate could serve as an electron donor for dechlorination by the H2-PCE culture, and it did not produce CH4 or acetate from H2-CO2 or methanol, indicating the absence of methanogenic and acetogenic bacteria. Microscopic observatios of the pruified H2-PCE culture showed only two major morphotypes: irregular cocci and small rods.     

Characterization of hydrogenase and reductive dehalogenase activities of Dehalococcoides ethenogenes strain 195

Dehalococcoides ethenogenes strain 195 reductively dechlorinates tetrachloroethene (PCE) and trichloroethene (TCE) to vinyl chloride and ethene using H2 as an electron donor. PCE- and TCE-reductive dehalogenase (RD) activities were mainly membrane associated, whereas only about 20% of the hydrogenase activity was membrane associated. Experiments with methyl viologen (MV) were consistent with a periplasmic location for the RDs or a component feeding electrons to them. The protonophore uncoupler tetrachlorosalicylanilide did not inhibit reductive dechlorination in cells incubated with H2 and PCE and partially restored activity in cells incubated with the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. Benzyl viologen or diquat (Eo' approximately -360 mV) supported reductive dechlorination of PCE or TCE at rates comparable to MV (-450 mV) in cell extracts.     

Complete detoxification of vinyl chloride by an anaerobic enrichment culture and identification of the reductively dechlorinating population as a Dehalococcoides species

A major obstacle in the implementation of the reductive dechlorination process at chloroethene-contaminated sites is the accumulation of the intermediate vinyl chloride (VC), a proven human carcinogen. To shed light on the microbiology involved in the final critical dechlorination step, a sediment-free, nonmethanogenic, VC-dechlorinating enrichment culture was derived from tetrachloroethene (PCE)-to-ethene-dechlorinating microcosms established with material from the chloroethene-contaminated Bachman Road site aquifer in Oscoda, Mich. After 40 consecutive transfers in defined, reduced mineral salts medium amended with VC, the culture lost the ability to use PCE and trichloroethene (TCE) as metabolic electron acceptors. PCE and TCE dechlorination occurred in the presence of VC, presumably in a cometabolic process. Enrichment cultures supplied with lactate or pyruvate as electron donor dechlorinated VC to ethene at rates up to 54 micromol liter(-1)day(-1), and dichloroethenes (DCEs) were dechlorinated at about 50% of this rate. The half-saturation constant (K(S)) for VC was 5.8 microM, which was about one-third lower than the concentrations determined for cis-DCE and trans-DCE. Similar VC dechlorination rates were observed at temperatures between 22 and 30 degrees C, and negligible dechlorination occurred at 4 and 35 degrees C. Reductive dechlorination in medium amended with ampicillin was strictly dependent on H(2) as electron donor. VC-dechlorinating cultures consumed H(2) to threshold concentrations of 0.12 ppm by volume. 16S rRNA gene-based tools identified a Dehalococcoides population, and Dehalococcoides-targeted quantitative real-time PCR confirmed VC-dependent growth of this population. These findings demonstrate that Dehalococcoides populations exist that use DCEs and VC but not PCE or TCE as metabolic electron acceptors.     

Complete biological reductive transformation of tetrachloroethene to ethane.

Reductive dechlorination of tetrachloroethene (perchloroethylene; PCE) was observed at 20 degrees C in a fixed-bed column, filled with a mixture (3:1) of anaerobic sediment from the Rhine river and anaerobic granular sludge. In the presence of lactate (1 mM) as an electron donor, 9 microM PCE was dechlorinated to ethene. Ethene was further reduced to ethane. Mass balances demonstrated an almost complete conversion (95 to 98%), with no chlorinated compounds remaining (less than 0.5 micrograms/liter). When the temperature was lowered to 10 degrees C, an adaptation of 2 weeks was necessary to obtain the same performance as at 20 degrees C. Dechlorination by column material to ethene, followed by a slow ethane production, could also be achieved in batch cultures. Ethane was not formed in the presence of bromoethanesulfonic acid, an inhibitor of methanogenesis. The high dechlorination rate (3.7 mumol.l-1.h-1), even at low temperatures and considerable PCE concentrations, together with the absence of chlorinated end products, makes reductive dechlorination an attractive method for removal of PCE in bioremediation processes.     

Complete biological reductive transformation of tetrachloroethene to ethane.

Reductive dechlorination of tetrachloroethene (perchloroethylene; PCE) was observed at 20 degrees C in a fixed-bed column, filled with a mixture (3:1) of anaerobic sediment from the Rhine river and anaerobic granular sludge. In the presence of lactate (1 mM) as an electron donor, 9 microM PCE was dechlorinated to ethene. Ethene was further reduced to ethane. Mass balances demonstrated an almost complete conversion (95 to 98%), with no chlorinated compounds remaining (less than 0.5 micrograms/liter). When the temperature was lowered to 10 degrees C, an adaptation of 2 weeks was necessary to obtain the same performance as at 20 degrees C. Dechlorination by column material to ethene, followed by a slow ethane production, could also be achieved in batch cultures. Ethane was not formed in the presence of bromoethanesulfonic acid, an inhibitor of methanogenesis. The high dechlorination rate (3.7 mumol.l-1.h-1), even at low temperatures and considerable PCE concentrations, together with the absence of chlorinated end products, makes reductive dechlorination an attractive method for removal of PCE in bioremediation processes.     

Reductive dechlorination of chlorinated ethene DNAPLs by a culture enriched from contaminated groundwater

A microbial culture enriched from a trichloroethene-contaminated groundwater aquifer reductively dechlorinated trichloroethene (TCE) and tetrachloroethene (PCE) to ethene. Initial PCE dechlorination rate studies indicated a first-order dependence with respect to substrate at low PCE concentrations, and a zero-order dependence at high concentrations. Studies of TCE and vinyl chloride (VC) dechlorination indicated a first-order dependence at all substrate concentrations. VC had little or no effect on the initial rate of TCE dechlorination. With subsaturating concentrations of chlorinated ethenes, nearly stoichiometric amounts of the toxic intermediate vinyl chloride accumulated prior to its dechlorination to ethene. In contrast, under saturating conditions, in which a dense, nonaqueous-phase liquid existed in equilibrium with the aqueous phase, the chlorinated ethene was dechlorinated to ethene, at a rapid rate, with the accumulation of relatively small amounts of chlorinated intermediates.     

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.     

Influence of vitamin B12 and cocultures on the growth of Dehalococcoides isolates in defined medium

Bacteria belonging to the genus Dehalococcoides play a key role in the complete detoxification of chloroethenes as these organisms are the only microbes known to be capable of dechlorination beyond dichloroethenes to vinyl chloride (VC) and ethene. However, Dehalococcoides strains usually grow slowly with a doubling time of 1 to 2 days and have complex nutritional requirements. Here we describe the growth of Dehalococcoides ethenogenes 195 in a defined mineral salts medium, improved growth of strain 195 when the medium was amended with high concentrations of vitamin B(12), and a strategy for maintaining Dehalococcoides strains on lactate by growing them in consortia. Although strain 195 could grow in defined medium spiked with approximately 0.5 mM trichloroethene (TCE) and 0.001 mg/liter vitamin B(12), the TCE dechlorination and cellular growth rates doubled when the vitamin B(12) concentration was increased 25-fold to 0.025 mg/liter. In addition, the final ratios of ethene to VC increased when the higher vitamin concentration was used, which reflected the key role that cobalamin plays in dechlorination reactions. No further improvement in dechlorination or growth was observed when the vitamin B(12) concentration was increased to more than 0.025 mg/liter. In defined consortia containing strain 195 along with Desulfovibrio desulfuricans and/or Acetobacterium woodii and containing lactate as the electron donor, tetrachloroethene ( approximately 0.4 mM) was completely dechlorinated to VC and ethene and there was concomitant growth of Dehalococcoides cells. In the cultures that also contained D. desulfuricans and/or A. woodii, strain 195 cells grew to densities that were 1.5 times greater than the densities obtained when the isolate was grown alone. The ratio of ethene to VC was highest in the presence of A. woodii, an organism that generates cobalamin de novo during metabolism. These findings demonstrate that the growth of D. ethenogenes strain 195 in defined medium can be optimized by providing high concentrations of vitamin B(12) and that this strain can be grown to higher densities in cocultures with fermenters that convert lactate to generate the required hydrogen and acetate and that may enhance the availability of vitamin B(12).     

Hydrogen as an electron donor for dechlorination of tetrachloroethene by an anaerobic mixed culture.

Hydrogen served as an electron donor in the reductive dechlorination of tetrachloroethene to vinyl chloride and ethene over periods of 14 to 40 days in anaerobic enrichment cultures; however, sustained dechlorination for more extended periods required the addition of filtered supernatant from a methanol-fed culture. This result suggests a nutritional dependency of hydrogen-utilizing dechlorinators on the metabolic products of other organisms in the more diverse, methanol-fed system. Vancomycin, an inhibitor of cell wall synthesis in eubacteria, was found to inhibit acetogenesis when added at 100 mg/liter to both methanol-fed and hydrogen-fed cultures. The effect of vancomycin on dechlorination was more complex. Methanol could not sustain dechlorination when vancomycin inhibited acetogenesis, while hydrogen could. These results are consistent with a model in which hydrogen is the electron donor directly used for dechlorination by organisms resistant to vancomycin and with the hypothesis that the role of acetogens in methanol-fed cultures is to metabolize a portion of the methanol to hydrogen. Methanol and other substrates shown to support dechlorination in pure and mixed cultures may merely serve as precursors for the formation of an intermediate hydrogen pool. This hypothesis suggests that, for bioremediation of high levels of tetrachloroethene, electron donors that cause the production of a large hydrogen pool should be selected or methods that directly use H2 should be devised.     

Hydrogen as an electron donor for dechlorination of tetrachloroethene by an anaerobic mixed culture.

Hydrogen served as an electron donor in the reductive dechlorination of tetrachloroethene to vinyl chloride and ethene over periods of 14 to 40 days in anaerobic enrichment cultures; however, sustained dechlorination for more extended periods required the addition of filtered supernatant from a methanol-fed culture. This result suggests a nutritional dependency of hydrogen-utilizing dechlorinators on the metabolic products of other organisms in the more diverse, methanol-fed system. Vancomycin, an inhibitor of cell wall synthesis in eubacteria, was found to inhibit acetogenesis when added at 100 mg/liter to both methanol-fed and hydrogen-fed cultures. The effect of vancomycin on dechlorination was more complex. Methanol could not sustain dechlorination when vancomycin inhibited acetogenesis, while hydrogen could. These results are consistent with a model in which hydrogen is the electron donor directly used for dechlorination by organisms resistant to vancomycin and with the hypothesis that the role of acetogens in methanol-fed cultures is to metabolize a portion of the methanol to hydrogen. Methanol and other substrates shown to support dechlorination in pure and mixed cultures may merely serve as precursors for the formation of an intermediate hydrogen pool. This hypothesis suggests that, for bioremediation of high levels of tetrachloroethene, electron donors that cause the production of a large hydrogen pool should be selected or methods that directly use H2 should be devised.     

Characterization of Hydrogenase and Reductive Dehalogenase Activities of Dehalococcoides ethenogenes Strain 195

Dehalococcoides ethenogenes strain 195 reductively dechlorinates tetrachloroethene (PCE) and trichloroethene (TCE) to vinyl chloride and ethene using H₂ as an electron donor. PCE- and TCE-reductive dehalogenase (RD) activities were mainly membrane associated, whereas only about 20% of the hydrogenase activity was membrane associated. Experiments with methyl viologen (MV) were consistent with a periplasmic location for the RDs or a component feeding electrons to them. The protonophore uncoupler tetrachlorosalicylanilide did not inhibit reductive dechlorination in cells incubated with H₂ and PCE and partially restored activity in cells incubated with the ATPase inhibitor N,N′-dicyclohexylcarbodiimide. Benzyl viologen or diquat (E^o′ ≈ −360 mV) supported reductive dechlorination of PCE or TCE at rates comparable to MV (−450 mV) in cell extracts.     

Complete dechlorination of tetrachloroethene to ethene in presence of methanogenesis and acetogenesis by an anaerobic sediment microcosm

An anaerobic consortium taken from brackish sediments, enriched byPCE/CH₃OH sequential feeding, was capable of completely dechlorinating tetrachloroethene(PCE) to ethene (ETH). In batch experiments, PCE (0.5 mM) was dechlorinated to ethene (ETH) in approximately 75 h with either CH₃OH or H₂ as the electron donor. When VC (0.5 mM) was added instead of PCE it was dechlorinated without any initial lag by the PCE/CH₃OHenriched consortium, although at a lower dechlorination rate. In batch tests H₂ could readilyreplace CH₃OH for supporting PCE dechlorination, with a similar PCE dechlorination rate andproduct distribution with respect to those observed with methanol. This indicates that H₂ productionduring CH₃OH fermentation was not the rate-limiting step of PCE or VC dechlorination.Acetogenesis was the predominant activity when methanol was present. A remarkable homoacetogenicactivity was also observed when hydrogen was supplied instead of methanol.     

Comparative study of methanol, butyrate, and hydrogen as electron donors for long-term dechlorination of tetrachloroethene in mixed anerobic cultures

This study examined the ability of different electron donors (i.e., hydrogen, methanol, butyrate, and yeast extract) to sustain long-term (500 days) reductive dechlorination of tetrachloroethene (PCE) in anerobic fill-and-draw bioreactors operated at 3:1 donor:PCE ratio (defined on a total-oxidation basis for the donor). Initially (i.e., until approximately day 80), the H(2)-fed bioreactor showed the best ability to completely dechlorinate the dosed PCE (0.5 mmol/L) to ethene whereas, in the presence of methanol, butyric acid or no electron donor added (but low-level yeast extract), dechlorination was limited by the fermentation of the organic substrates and in turn by H(2) availability. As the study progressed, the H(2)-fed reactor experienced a diminishing ability to dechlorinate, while more stable dechlorinating activity was maintained in the reactors that were fed organic donors. The initial diminished ability of the H(2)-fed reactor to dechlorinate (after about 100 days), could be partially explained in terms of increased competition for H(2) between dechlorinators and methanogens, whereas other factors such as growth-factor limitation and/or accumulation of toxic and/or inhibitory metabolites were shown to play a role for longer incubation periods (over 500 days). In spite of decreasing activity with time, the H(2)-fed reactor proved to be the most effective in PCE dechlorination: after about 500 days, more than 65% of the added PCE was dechlorinated to ethene in the H(2)-fed reactor, versus 36%, 22%, and <1% in the methanol-fed, butyrate-fed, and control reactors, respectively.     

Reductive dechlorination of tetrachloroethene to ethene by a two-component enzyme pathway

Two membrane-bound, reductive dehalogenases that constitute a novel pathway for complete dechlorination of tetrachloroethene (perchloroethylene [PCE]) to ethene were partially purified from an anaerobic microbial enrichment culture containing Dehalococcoides ethenogenes 195. When titanium (III) citrate and methyl viologen were used as reductants, PCE-reductive dehalogenase (PCE-RDase) (51 kDa) dechlorinated PCE to trichloroethene (TCE) at a rate of 20 micromol/min/mg of protein. TCE-reductive dehalogenase (TCE-RDase) (61 kDa) dechlorinated TCE to ethene. TCE, cis-1,2-dichloroethene, and 1,1-dichloroethene were dechlorinated at similar rates, 8 to 12 micromol/min/mg of protein. Vinyl chloride and trans-1,2-dichloroethene were degraded at rates which were approximately 2 orders of magnitude lower. The light-reversible inhibition of TCE-RDase by iodopropane and the light-reversible inhibition of PCE-RDase by iodoethane suggest that both of these dehalogenases contain Co(I) corrinoid cofactors. Isolation and characterization of these novel bacterial enzymes provided further insight into the catalytic mechanisms of biological reductive dehalogenation.     

Adaptation of a constructed wetland to simultaneous treatment of monochlorobenzene and perchloroethene

Mixed groundwater contaminations by chlorinated volatile organic compounds (VOC) cause environmental hazards if contaminated groundwater discharges into surface waters and river floodplains. Constructed wetlands (CW) or engineered natural wetlands provide a promising technology for the protection of sensitive water bodies. We adapted a constructed wetland able to treat monochlorobenzene (MCB) contaminated groundwater to a mixture of MCB and tetrachloroethene (PCE), representing low and high chlorinated model VOC. Simultaneous treatment of both compounds was efficient after an adaptation time of 2 1/2 years. Removal of MCB was temporarily impaired by PCE addition, but after adaptation a MCB concentration decrease of up to 64% (55.3 micromol L(-1)) was observed. Oxygen availability in the rhizosphere was relatively low, leading to sub-optimal MCB elimination but providing also appropriate conditions for PCE dechlorination. PCE and metabolites concentration patterns indicated a very slow system adaptation. However, under steady state conditions complete removal of PCE inflow concentrations of 10-15 micromol L(-1) was achieved with negligible concentrations of chlorinated metabolites in the outflow. Recovery of total dechlorination metabolite loads corresponding to 100%, and ethene loads corresponding to 30% of the PCE inflow load provided evidence for complete reductive dechlorination, corroborated by the detection of Dehalococcoides sp.     

Quantitative PCR confirms purity of strain GT, a novel trichloroethene-to-ethene-respiring Dehalococcoides isolate

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 micromol liter-1 day-1, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76+/-0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.     

Reductive dechlorination of chlorinated ethenes and 1, 2-dichloroethane by "Dehalococcoides ethenogenes" 195.

"Dehalococcoides ethenogenes" 195 can reductively dechlorinate tetrachloroethene (PCE) completely to ethene (ETH). When PCE-grown strain 195 was transferred (2% [vol/vol] inoculum) into growth medium amended with trichloroethene (TCE), cis-dichloroethene (DCE), 1,1-DCE, or 1,2-dichloroethane (DCA) as an electron acceptor, these chlorinated compounds were consumed at increasing rates over time, which indicated that growth occurred. Moreover, the number of cells increased when TCE, 1,1-DCE, or DCA was present. PCE, TCE, 1,1-DCE, and cis-DCE were converted mainly to vinyl chloride (VC) and then to ETH, while DCA was converted to ca. 99% ETH and 1% VC. cis-DCE was used at lower rates than PCE, TCE, 1,1-DCE, or DCA was used. When PCE-grown cultures were transferred to media containing VC or trans-DCE, products accumulated slowly, and there was no increase in the rate, which indicated that these two compounds did not support growth. When the intermediates in PCE dechlorination by strain 195 were monitored, TCE was detected first, followed by cis-DCE. After a lag, VC, 1,1-DCE, and trans-DCE accumulated, which is consistent with the hypothesis that cis-DCE is the precursor of these compounds. Both cis-DCE and 1,1-DCE were eventually consumed, and both of these compounds could be considered intermediates in PCE dechlorination, whereas the small amount of trans-DCE that was produced persisted. Cultures grown on TCE, 1,1-DCE, or DCA could immediately dechlorinate PCE, which indicated that PCE reductive dehalogenase activity was constitutive when these electron acceptors were used.     

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.     

Use of Silicate Minerals for pH Control during Reductive Dechlorination of Chloroethenes in Batch Cultures of Different Microbial Consortia

In chloroethene-contaminated sites undergoing in situ bioremediation, groundwater acidification is a frequent problem in the source zone, and buffering strategies have to be implemented to maintain the pH in the neutral range. An alternative to conventional soluble buffers is silicate mineral particles as a long-term source of alkalinity. In previous studies, the buffering potentials of these minerals have been evaluated based on abiotic dissolution tests and geochemical modeling. In the present study, the buffering potentials of four silicate minerals (andradite, diopside, fayalite, and forsterite) were tested in batch cultures amended with tetrachloroethene (PCE) and inoculated with different organohalide-respiring consortia. Another objective of this study was to determine the influence of pH on the different steps of PCE dechlorination. The consortia showed significant differences in sensitivities toward acidic pH for the different dechlorination steps. Molecular analysis indicated that Dehalococcoides spp. that were present in all consortia were the most pH-sensitive organohalide-respiring guild members compared to Sulfurospirillum spp. and Dehalobacter spp. In batch cultures with silicate mineral particles as pH-buffering agents, all four minerals tested were able to maintain the pH in the appropriate range for reductive dechlorination of chloroethenes. However, complete dechlorination to ethene was observed only with forsterite, diopside, and fayalite. Dissolution of andradite increased the redox potential and did not allow dechlorination. With forsterite, diopside, and fayalite, dechlorination to ethene was observed but at much lower rates for the last two dechlorination steps than with the positive control. This indicated an inhibition effect of silicate minerals and/or their dissolution products on reductive dechlorination of cis-dichloroethene and vinyl chloride. Hence, despite the proven pH-buffering potential of silicate minerals, compatibility with the bacterial community involved in in situ bioremediation has to be carefully evaluated prior to their use for pH control at a specific site.