Anaerobic Bioremediation of Groundwater Containing a Mixture of 1,1,2,2-Tetrachloroethane and Chloroethenes

This study investigated the biotransformation pathways of 1,1,2,2-tetrachloroethane (1,1,2,2-TeCA) in the presence of chloroethenes (i.e. tetrachloroethene, PCE; trichloroethene, TCE) in anaerobic microcosms constructed with subsurface soil and groundwater from a contaminated site. When amended with yeast extract, lactate, butyrate, or H₂ and acetate, 1,1,2,2-TeCA was initially dechlorinated via both hydrogenolysis to 1,1,2-trichloroethane (1,1,2-TCA) (major pathway) and dichloroelimination to dichloroethenes (DCEs) (minor pathway), with both reactions occurring under sulfidogenic conditions. In the presence of only H₂, the hydrogenolysis of 1,1,2,2-TeCA to 1,1,2-TCA apparently required the presence of acetate to occur. Once formed, 1,1,2-TCA was degraded predominantly via dichloroelimination to vinyl chloride (VC). Ultimately, chloroethanes were converted to chloroethenes (mainly VC and DCEs) which persisted in the microcosms for very long periods along with PCE and TCE originally present in the groundwater. Hydrogenolysis of chloroethenes occurred only after highly reducing methanogenic conditions were established. However, substantial conversion to ethene (ETH) was observed only in microcosms amended with yeast extract (200 mg/l), suggesting that groundwater lacked some nutritional factors which were likely provided to dechlorinating microorganisms by this complex organic substrate. Bioaugmentation with an H₂-utilizing PCE-dechlorinating Dehalococcoides spp. -containing culture resulted in the conversion of 1,1,2,2-TeCA, PCE and TCE to ETH and VC. No chloroethanes accumulated during degradation suggesting that 1,1,2,2-TeCA was degraded through initial dichloroelimination into DCEs and then typical hydrogenolysis into ETH and VC.     

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 μmol liter⁻¹day⁻¹, and dichloroethenes (DCEs) were dechlorinated at about 50% of this rate. The half-saturation constant (K_S) for VC was 5.8 μM, 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°C, and negligible dechlorination occurred at 4 and 35°C. Reductive dechlorination in medium amended with ampicillin was strictly dependent on H₂ as electron donor. VC-dechlorinating cultures consumed H₂ 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.     

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.     

Complete reductive dechlorination of tetrachloroethene to ethene by anaerobic microbial enrichment culture developed from sediment

A mixed, anaerobic microbial enrichment culture, AMEC-4P, was developed that uses lactate as the electron donor for the reductive dechlorination of tetrachloroethene (PCE) to ethene. AMEC-4P consistently and completely converted 2 mM PCE to cis-1,2-dichloroethene (cis-DCE) within 13 days, and the intermediate, cis-DCE, was then completely dechlorinated to ethene after 130 days. Dechlorination rates for PCE to cis-DCE, cis-DCE to VC, and VC to ethene were 243, 27, and 41 μmol/l/day, respectively. Geobacter lovleyi and a Dehalococcoides sp. were identified from their 16S rRNA sequences to be the dominant phylotypes in AMEC-4P.     

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 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.     

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.     

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.     

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 B₁₂, and a strategy for maintaining Dehalococcoides strains on lactate by growing them in consortia. Although strain 195 could grow in defined medium spiked with ~0.5 mM trichloroethene (TCE) and 0.001 mg/liter vitamin B₁₂, the TCE dechlorination and cellular growth rates doubled when the vitamin B₁₂ 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 B₁₂ 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 (~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 B₁₂ 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 B₁₂.     

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.     

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.     

Reductive dechlorination of perchloroethylene and the role of methanogens

Abstract Perchloroethylene (PCE) was reductively dechlorinated to trichloroethylene in a 10% anaerobic sewage sludge. About 80% of the initially added PCE (300 nmol) was dechlorinated within three weeks. The calculated rates were 250 nM and 445 nM · day⁻¹ during the first and second weeks of incubation, respectively. The depletion of PCE varied in sludges obtained from different sources.
The role of methanogenesis in the dechlorination of PCE was evaluated by inhibiting the methanogens by addition of bromoethane sulfonic acid, a potent methanogenic inhibitor. Dechlorination of PCE was significantly inhibited in sludges amended with the inhibitor. Almost 41–48% less PCE was dechlorinated in sludges containing 5 mM BESA, indicating a relation between the two processes (methanogenesis and dechlorination). Direct proof that methanogens can transform chlorinated aliphatic compounds was obtained using axenic cultures of acetate-cleaving methanogens. Methanosarcina sp , originally isolated from a chlorophenol degrading consortium, showed significantly higher dechlorinating activity as compared to Ms. mazei . Based on these studies and other recently reported observations, it appears that methanogens/methanogenesis play an important role in the anaerobic dechlorination of chlorinated aliphatics such as PCE.     

Kinetics of chlorinated ethylene dehalogenation under methanogenic conditions

Kinetics were determined for methanogenic activity and chlorinated ethylene dehalogenation by a methanol-enriched, anaerobic sediment consortium. The culture reductively dechlorinated perchloroethylene (PCE) to trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), vinylchloride (VC), and ethylene and ethane. The absence : of methanol or the addition of 2-bromoethanesulfonic. acid in the presence of methanol suppressed both methanogenic activity and dechlorination. In contrast, acetate production continued in the presence of 2-bromoethanesulfonic acid. These results suggest that dechlorination was strongly linked to methane formation and not to acetate production. A kinetic model, developed to describe both methanogenesis and dechlorination, successfully predicted experimentally measured concentrations of biomass, methane, substrate, and chlorinated ethylenes. The average maximum specific dehalogenation rates for PCE, TCE, 1,1-DCE, and VC were 0.9 +/- 0.6, 0.4 +/- 0.1, 12 +/- 0.1, and 2.5 +/- 1.7 mumol contaminant/ g. DW/day, respectively. This pattern for dechlorination rates is distinctly different than that reported for transition metal cofactors, where rates drop by approximately one order of magnitude as each successive chlorine is removed. The experimental results and kinetic analysis suggest that it will be impractical to targeting methanol consuming methanogenic organisms for in situ ground-water restoration. (c) 1995 John Wiley & Sons, Inc.     

Monitoring Redox Conditions with Flow-Based and Fiber Optic Sensors Based on Redox Indicators: Application to Reductive Dehalogenation in a Bioaugmented Soil Column

Redox indicators were employed to monitor redox status in a bioaugmented, sediment-packed column during the dechlorination of tetrachloroethene (PCE) to ethene (ETH). The speciation of the indicators thionine and cresyl violet, immobilized on transparent films, was spectrometrically monitored with a flow sensor based on circulating the column solution through a specially constructed flow cell placed in a conventional spectrometer. A fiber optic redox probe based on immobilized azure C was constructed. A 75-cm column with 4 sets of ports along the column axis at regular intervals was constructed and packed with aquifer material. These ports enabled sampling to determine concentrations of chlorinated ethene species and allowed for in situ or non-invasive monitoring of redox conditions with negligible O ₂ contamination. The flow sensor and fiber optic sensor showed similar responses to redox conditions in the column. After 60 days, complete conversion of PCE to ETH occurred by the end of the column and the redox level indicated by the indicators was consistent throughout the column. Significant formation of vinyl chloride or ETH was observed only after significant reduction of cresyl violet.     

Dechlorination of PCE by Mixed Methanogenic Cultures Using Hollow-Fiber Membranes

The study investigated the use of hollow-fiber membranes for hydrogen (H₂) delivery to support the biological reductive dechlorination of tetrachloroethene (PCE) Two experiments were performed in which H₂ was supplied through membranes placed in stirred batch reactors containing a mixed methanogenic/dechlorinating culture and PCE (≤10?µM. Reductive dechlorination of PCE to cis-dichloroethene was sustained in the reactors receiving H₂ (1% H₂ and 50% H₂), while negligible dechlorination was observed in control reactors (100% N₂). The 1%-H₂-fed reactor outperformed the 50%-H₂-fed reactor in the first experiment. However, the dechlorinating performance in the two reactors was similar in the second experiment. Despite relatively high H₂ concentrations (4.6 to 178?µM) that led to H₂ consumption (and CH₄ production) by methanogens, dechlorination was effectively maintained for the duration of the experiments (35 to 62 days). The results of this study are significant in that dechlorination was sustained in a minimal medium by membrane-delivered H₂. Dechlorination was also maintained at aqueous H₂ concentrations that exceeded the thermodynamic thresholds for not only dechlorination (<0.1 to 2?nM, but also methanogenesis (～10?n_M) and homoacetogenesis (94 to 400?n_M. The results of these experiments suggest that membranes are a promising H₂ delivery technology for stimulating the bioremediation of chlorinated ethene-contaminated aquifers.     

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.     

Methanosarcina spp. Drive Vinyl Chloride Dechlorination via Interspecies Hydrogen Transfer

Two highly enriched cultures containing Dehalococcoides spp. were used to study the effect of aceticlastic methanogens on reductive vinyl chloride (VC) dechlorination. In terms of aceticlastic methanogens, one culture was dominated by Methanosaeta, while the other culture was dominated by Methanosarcina, as determined by fluorescence in situ hybridization. Cultures amended with 2-bromoethanesulfonate (BES), an efficient inhibitor of methanogens, exhibited slow VC dechlorination when grown on acetate and VC. Methanogenic cultures dominated by Methanosaeta had no impact on dechlorination rates, compared to BES-amended controls. In contrast, methanogenic cultures dominated by Methanosarcina displayed up to sevenfold-higher rates of VC dechlorination than their BES-amended counterparts. Methanosarcina-dominated cultures converted a higher percentage of [2-¹⁴C]acetate to ¹⁴CO₂ when concomitant VC dechlorination took place, compared to nondechlorinating controls. Respiratory indices increased from 0.12 in nondechlorinating cultures to 0.51 in actively dechlorinating cultures. During VC dechlorination, aqueous hydrogen (H₂) concentrations dropped to 0.3 to 0.5 nM. However, upon complete VC consumption, H₂ levels increased by a factor of 10 to 100, indicating active hydrogen production from acetate oxidation. This process was thermodynamically favorable by means of the extremely low H₂ levels during dechlorination. VC degradation in nonmethanogenic cultures was not inhibited by BES but was limited by the availability of H₂ as electron donor, in cultures both with and without BES. These findings all indicate that Methanosarcina (but not Methanosaeta), while cleaving acetate to methane, simultaneously oxidizes acetate to CO₂ plus H₂, driving hydrogenotrophic dehalorespiration of VC to ethene by Dehalococcoides.     

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.     

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 μmol 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.