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.     

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.     

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.     

Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes

Many reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediates cis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinating Geobacter and several Dehalococcoides strains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase from Geobacter was only detected transiently at the beginning of TCE dechlorination. The Dehalococcoides RDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. The Dehalococcoides RDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity. trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains of Dehalococcoides as a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities.     

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.     

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.     

Frequent concomitant presence of Desulfitobacterium spp. and "Dehalococcoides" spp. in chloroethene-dechlorinating microbial communities

The presence of chloroethene dechlorination activity as well as several bacterial genera containing mainly organohalide-respiring members was investigated in 34 environmental samples from 18 different sites. Cultures inoculated with these environmental samples on tetrachloroethene and amended weekly with a seven organic electron donor mixture resulted in 11 enrichments with cis-DCE, ten with VC, and 11 with ethene as dechlorination end product, and only two where no dechlorination was observed. “Dehalococcoides” spp. and Desulfitobacterium spp. were detected in the majority of the environmental samples independently of the dechlorination end product formed. The concomitant presence of Dehalococcoides spp. and Desulfitobacterium spp. in the majority of the enrichments suggested that chloroethene dechlorination was probably the result of catalysis by at least two organohalide-respiring genera either in parallel or by stepwise catalysis. A more detailed study of one enrichment on cis-DCE suggested that in this culture Desulfitobacterium spp. as well as Dehalococcoides spp. dechlorinated cis-DCE whereas dechlorination of VC was only catalyzed by the latter.     

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.     

Discrimination of Multiple Dehalococcoides Strains in a Trichloroethene Enrichment by Quantification of Their Reductive Dehalogenase Genes

While many anaerobic microbial communities are capable of reductively dechlorinating tetrachloroethene (PCE) and trichloroethene (TCE) to dichloroethene (DCE), vinyl chloride (VC), and finally ethene, the accumulation of the highly toxic intermediates, cis-DCE (cDCE) and VC, presents a challenge for bioremediation processes. Members of the genus Dehalococcoides are apparently solely responsible for dechlorination beyond DCE, but isolates of Dehalococcoides each metabolize only a subset of PCE dechlorination intermediates and the interactions among distinct Dehalococcoides strains that result in complete dechlorination are not well understood. Here we apply quantitative PCR to 16S rRNA and reductase gene sequences to discriminate and track Dehalococcoides strains in a TCE enrichment derived from soil taken from the Alameda Naval Air Station (ANAS) using a four-gene plasmid standard. This standard increased experimental accuracy such that 16S rRNA and summed reductase gene copy numbers matched to within 10%. The ANAS culture was found to contain only a single Dehalococcoides 16S rRNA gene sequence, matching that of D. ethenogenes 195, but both the vcrA and tceA reductive dehalogenase genes. Quantities of these two genes in the enrichment summed to the quantity of the Dehalococcoides 16S rRNA gene. Further, between ANAS subcultures enriched on TCE, cDCE, or VC, the relative copy number of the two dehalogenases shifted 14-fold, indicating that the genes are present in two different Dehalococcoides strains. Comparison of cell yields in VC-, cDCE-, and TCE-enriched subcultures suggests that the tceA-containing strain is responsible for nearly all of the TCE and cDCE metabolism in ANAS, whereas the vcrA-containing strain is responsible for all of the VC metabolism.     

Effects of bioaugmentation on enhanced reductive dechlorination of 1,1,1-trichloroethane in groundwater: a comparison of three sites

Microcosm studies investigated the effects of bioaugmentation with a mixed Dehalococcoides (Dhc)/Dehalobacter (Dhb) culture on biological enhanced reductive dechlorination for treatment of 1,1,1-trichloroethane (TCA) and chloroethenes in groundwater at three Danish sites. Microcosms were amended with lactate as electron donor and monitored over 600 days. Experimental variables included bioaugmentation, TCA concentration, and presence/absence of chloroethenes. Bioaugmented microcosms received a mixture of the Dhc culture KB-1 and Dhb culture ACT-3. To investigate effects of substrate concentration, microcosms were amended with various concentrations of chloroethanes (TCA or monochloroethane [CA]) and/or chloroethenes (tetrachloroethene [PCE], trichloroethene [TCE], or 1,1-dichloroethene [1,1-DCE]). Results showed that combined electron donor addition and bioaugmentation stimulated dechlorination of TCA and 1,1-dichloroethane (1,1-DCA) to CA, and dechlorination of PCE, TCE, 1,1-DCE and cDCE to ethane. Dechlorination of CA was not observed. Bioaugmentation improved the rate and extent of TCA and 1,1-DCA dechlorination at two sites, but did not accelerate dechlorination at a third site where geochemical conditions were reducing and Dhc and Dhb were indigenous. TCA at initial concentrations of 5 mg/L inhibited (i.e., slowed the rate of) TCA dechlorination, TCE dechlorination, donor fermentation, and methanogenesis. 1 mg/L TCA did not inhibit dechlorination of TCA, TCE or cDCE. Moreover, complete dechlorination of PCE to ethene was observed in the presence of 3.2 mg/L TCA. In contrast to some prior reports, these studies indicate that low part-per million levels of TCA (<3 mg/L) in aquifer systems do not inhibit dechlorination of PCE or TCE to ethene. In addition, the results show that co-bioaugmentation with Dhc and Dhb cultures can be an effective strategy for accelerating treatment of chloroethane/chloroethene mixtures in groundwater, with the exception that all currently known Dhc and Dhb cultures cannot treat CA.     

Isolation and Characterization of “Dehalococcoides” sp. Strain MB,Which Dechlorinates Tetrachloroethene to trans-1,2-Dichloroethene

In an attempt to understand the microorganisms involved in the generation of trans-1,2-dichloroethene (trans-DCE), pure-culture “Dehalococcoides” sp. strain MB was isolated from environmental sediments. In contrast to currently known tetrachloroethene (PCE)- or trichloroethene (TCE)-dechlorinating pure cultures, which generate cis-DCE as the predominant product, Dehalococcoides sp. strain MB reductively dechlorinates PCE to trans-DCE and cis-DCE at a ratio of 7.3 (±0.4):1. It utilizes H₂ as the sole electron donor and PCE or TCE as the electron acceptor during anaerobic respiration. Strain MB is a disc-shaped, nonmotile bacterium. Under an atomic force microscope, the cells appear singly or in pairs and are 1.0 μm in diameter and ∼150 nm in depth. The purity was confirmed by culture-based approaches and 16S rRNA gene-based analysis and was corroborated further by putative reductive dehalogenase (RDase) gene-based, quantitative real-time PCR. Although strain MB shares 100% 16S rRNA gene sequence identity with Dehalococcoides ethenogenes strain 195, these two strains possess different dechlorinating pathways. Microarray analysis revealed that 10 putative RDase genes present in strain 195 were also detected in strain MB. Successful cultivation of strain MB indicates that the biotic process could contribute significantly to the generation of trans-DCE in chloroethene-contaminated sites. It also enhances our understanding of the evolution of this unusual microbial group, Dehalococcoides species.Dehalorespiring bacteria play an important role in the transformation and detoxification of a wide range of halogenated compounds, e.g., chlorophenols, chloroethenes, chlorobenzenes, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs) (2, 4, 9, 14, 16, 17, 32, 35, 38). Among these compounds, the organic solvents tetrachloroethene (PCE) and trichloroethene (TCE) are suspected carcinogens that are found in soil and groundwater due to their extensive usage and improper disposal (6). The widespread PCE and TCE in the subsurface environment have driven intensive studies of anaerobic microbes capable of reductive dechlorination of chloroethenes (40). Over the last decade, at least 18 isolates, which belong to the genera Desulfitobacterium, Sulfurospirillum, Desulfomonile, Desulfuromonas, Geobacter, “Dehalococcoides,” and Dehalobacter, show reductive dechlorination of chlorinated ethenes (16, 40). In particular, most of these microbes produce cis-1,2-dichloroethene (cis-DCE) as the end product in the chloroethene-contaminated sites, whereas complete detoxification of PCE or TCE to ethene has been restricted only to members of the genus Dehalococcoides. Thus, the Dehalococcoides species have received considerable attention from the bioremediation community in the past decade.Several strains of Dehalococcoides species (e.g., 195, CBDB1, BAV1, and VS) have been sequenced for their whole genomes (24, 39). Their dechlorinating capabilities have also been well addressed through identification and quantification of the known chloroethene reductive dehalogenase (RDase) genes or expression of specific RDase genes (18, 21, 25, 41). In chloroethene-contaminated sites, the natural activities of single or multiple Dehalococcoides strains can lead either to more-toxic, mobile intermediates (e.g., cis- or trans-DCEs and vinyl chloride [VC]) via partial dechlorination of PCE/TCE or to harmless ethene by complete detoxification (10, 13, 15, 41). Many mixed cultures and pure isolates have been reported to produce cis-DCE or VC during PCE/TCE dechlorination processes (15, 40, 43). However, trans-DCE has been detected in more than one-third of the U.S. Environmental Protection Agency (EPA) superfund sites (3a). The source of trans-DCE production was thought to be an abiotic process; however, recently both trans-DCE generation and cis-DCE generation were reported to occur via microbial dechlorination.To date, microbes from either Dehalococcoides- or DF-1-containing mixed cultures have been reported to produce more trans- than cis-DCE, with a ratio of 1.2:1 to 3.5:1 in laboratory-scale studies (8, 10, 22, 31). For example, in a recent report by Kittelmann and Friedrich (22), trans-/cis-DCE at a ratio of 3.5:1 was generated in tidal flat sediment-containing microcosms with microbes closely related to Dehalococcoides sp. or DF-1-like microbes. Additionally, Griffin et al. identified Dehalococcoides species of the Pinellas subgroup in several enrichment cultures, which dechlorinated TCE (∼0.25 mM) to trans-DCE and cis-DCE at a ratio of ∼3:1 (10). There is no information available on the Dehalococcoides isolates that generate trans-DCE as the main end product. This also means a lack of information on the genomic contents of trans-DCE-producing bacteria. Therefore, finding microorganisms that produce trans-DCE in pure culture will be useful for the comprehensive characterization of this group of bacteria.The aim of this study was to isolate a PCE-to-trans-DCE-dechlorinating culture to facilitate the elucidation of trans-DCE formation during reductive dechlorination processes. Microarray analysis was conducted to compare the whole-genome contents of the new isolate and the well-characterized Dehalococcoides ethenogenes strain 195 (30). In addition, a coculture which consisted of the new isolate and TCE-to-cis-DCE-to-VC-dechlorinating Dehalococcoides sp. strain ANAS1 was explored to study the interaction, distribution, and function of the dechlorinators in the dechlorinating process.     

A highly purified enrichment culture couples the reductive dechlorination of tetrachloroethene to growth.

A microscopically pure enrichment culture of a gram-negative anaerobic bacterium, in the present article referred to as PER-K23, was isolated from an anaerobic packed-bed column in which tetrachloroethene (PCE) was reductively transformed to ethane via trichloroethene (TCE), cis-1,2-dichloroethene (cis-1,2-DCE), chloroethene, and ethene. PER-K23 catalyzes the dechlorination of PCE via TCE to cis-1,2-DCE and couples this reductive dechlorination to growth. H2 and formate were the only electron donors that supported growth with PCE or TCE as an electron acceptor. The culture did not grow in the absence of PCE or TCE. Neither O2, NO3-, NO2-, SO4(2-), SO3(2-), S2O3(2-), S, nor CO2 could replace PCE or TCE as an electron acceptor with H2 as an electron donor. Also, organic electron acceptors such as acetoin, acetol, dimethyl sulfoxide, fumarate, and trimethylamine N-oxide and chlorinated ethanes, DCEs, and chloroethene were not utilized. PER-K23 was not able to grow fermentatively on any of the organic compounds tested. Transferring the culture to a rich medium revealed that a contaminant was still present. Dechlorination was optimal between pH 6.8 and 7.6 and a temperature of 25 to 35 degrees C. H2 consumption was paralleled by chloride production, PCE degradation, cis-1,2-DCE formation, and growth of PER-K23. Electron balances showed that all electrons derived from H2 or formate consumption were recovered in dechlorination products and biomass. Exponential growth could be achieved only in gently shaken cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tetrachloroethene and 3-chlorobenzoate dechlorination activities are co-induced inDesulfomonile tiedjei DCB-1

Desulfomonile tiedjei, a strict anaerobe capable of reductively dechlorinating 3-chlorobenzoate, also dechlorinates tetrachloroethene and trichloroethene. It is not known, however, if the aryl and aliphatic dechlorination activities are catalyzed by the same enzymatic system. Cultures induced for 3-chlorobenzoate activity dechlorinated tetrachloroethene and trichloroethene to lower chlorinated products while uninduced parallel cultures did not dechlorinate either substrate. The observed rate of PCE dechlorination in induced cultures was 22 µmol h^–1 g protein^–1, which is considerably faster than previous rates obtained with defined cultures of this organism. These results show that both dechlorination activities are co-induced and therefore, that the dechlorination mechanisms may share at least some components.Abbreviations PCE tetrachloroethene - TCE trichloroethene - cis-DCE cis-dichloroethene - trans-DCE trans-dichloroethene - 3FBz 3-fluorobenzoate - 3ClBz 3-chlorobenzoate     

Correlation of Dehalococcoides 16S rRNA and Chloroethene-Reductive Dehalogenase Genes with Geochemical Conditions in Chloroethene-Contaminated Groundwater

Quantitative analysis of genes that code for Dehalococcoides 16S rRNA and chloroethene-reductive dehalogenases TceA, VcrA, and BvcA was done on groundwater sampled from 150 monitoring wells spread over 11 chlorinated ethene polluted European locations. Redundancy analysis was used to relate molecular data to geochemical conditions. Dehalococcoides 16S rRNA- and vinyl chloride (VC)-reductase genes were present at all tested locations in concentrations up to 10⁶ gene copies per ml of groundwater. However, differences between and also within locations were observed. Variation in Dehalococcoides 16S rRNA gene copy numbers were most strongly correlated to dissolved organic carbon concentration in groundwater and to conditions appropriate for biodegradation of chlorinated ethenes (U.S. Environmental Protection Agency score). In contrast, vcrA gene copy numbers correlated most significantly to VC and chlorinated ethene concentrations. Interestingly, bvcA and especially tceA were more correlated with oxidizing conditions. In groundwater microcosms, dechlorination of 1 mM VC was correlated to an increase of vcrA and/or bvcA gene copies by 2 to 4 orders of magnitude. Interestingly, in 34% of the monitoring wells and in 40% of the active microcosms, the amount of individual VC-reductase gene copies exceeded that of Dehalococcoides 16S rRNA gene copies. It is concluded that the geographical distribution of the genes was not homogeneous, depending on the geochemical conditions, whereby tceA and bvcA correlated to more oxidized conditions than Dehalococcoides 16S rRNA and vcrA. Because the variation in VC-reductase gene numbers was not directly correlated to variation in Dehalococcoides spp., VC-reductase genes are better monitoring parameters for VC dechlorination capacity than Dehalococcoides spp.Chlorinated ethenes, such as tetrachloroethene (PCE) and trichloroethene (TCE), are persistent groundwater pollutants (15, 22). Because these compounds are toxic and mobile in groundwater systems, they form a serious risk for human health and the environment. PCE and TCE can be dechlorinated by microorganisms under anaerobic conditions by reductive dehalogenation to dichloroethene (DCE), vinyl chloride (VC), and ethene (20). Bioremediation strategies for chloroethene-contaminated sites are often based on (stimulation of) reductive dechlorination of the chlorinated ethenes to ethene (7, 12, 14). In practice, reductive dechlorination of PCE and TCE can be incomplete, resulting in accumulation of DCE or VC. Since VC is much more mobile, toxic, and carcinogenic than PCE and TCE (9), monitoring and stimulation of VC dechlorination are essential steps in bioremediation strategies.Only members of Dehalococcoides spp. are known to be able to reductively dechlorinate VC. Therefore, 16S rRNA genes of these species are often used as molecular target to indicate and monitor DCE and VC dechlorination capacity at contaminated sites. However, previous studies showed different dechlorination capacities for individual Dehalococcoides species, and only a few strains are known to metabolically dechlorinate VC (6, 8, 10, 17, 21). As a consequence, 16S rRNA gene-based detection can lead to overestimation of VC dechlorination capacity. In contrast, although metabolic reductive dechlorination of VC has mostly been linked to Dehalococcoides spp., it cannot be excluded that other microbial species that perform this dechlorination exist. Genes coding for DCE and VC reductases may be exchangeable between different microbial species via horizontal gene transfer. This is plausible since it has been shown that the metabolic genes for VC dechlorination, vcrA and bvcA, have a different evolutionary history than most other Dehalococcoides genes (16). Consequently, Dehalococcoides 16S rRNA gene-based detection can also lead to underestimation of VC dechlorination capacity.To more precisely determine VC dechlorination capacity, genes directly involved in reductive dechlorination of VC should be used as a molecular target, in addition to Dehalococcoides 16S rRNA genes. A quantitative method was described to detect genes coding for VC-reductases VcrA and BvcA identified in Dehalococcoides sp. strains VS and GT and in Dehalococcoides sp. strain BAV1, respectively (10, 17, 21). Different studies showed direct correlation of vcrA and bvcA gene copy numbers with reductive dechlorination of VC in batch cultures, soil columns, and contaminated sites (2, 11, 19).Quantification of genes that encode VC-reductases can be a useful method to monitor reductive dechlorination of VC in chloroethene-contaminated groundwater during enhanced natural attenuation activities (4, 19). However, little is known about the presence, dispersion, and importance of specific dehalogenase genes in chlorinated ethene polluted groundwater and their correlation to biogeochemical conditions and reductive dechlorination.The objective of the present study was therefore to identify the relative importance of TCE-reductase gene tceA and VC-reductase genes vcrA and bvcA in chloroethene-polluted groundwater and to identify geochemical parameters that contribute to variation in copy numbers of these genes. To this end, groundwater of 150 monitoring wells from 11 European polluted sites was analyzed. Furthermore, microcosms with groundwater from 6 locations were started to test whether VC dechlorination is directly correlated to an increase of vcrA or bvcA genes.     

Reductive dechlorination of tetrachloroethene by a stepwise catalysis of different organohalide respiring bacteria and reductive dehalogenases

The enrichment culture SL2 dechlorinating tetrachloroethene (PCE) to ethene with strong trichloroethene (TCE) accumulation prior to cis-1,2-dichloroethene (cis-DCE) formation was analyzed for the presence of organohalide respiring bacteria and reductive dehalogenase genes (rdhA). Sulfurospirillum-affiliated bacteria were identified to be involved in PCE dechlorination to cis-DCE whereas “Dehalococcoides”-affiliated bacteria mainly dechlorinated cis-DCE to ethene. Two rdhA genes highly similar to tetrachloroethene reductive dehalogenase genes (pceA) of S. multivorans and S. halorespirans were present as well as an rdhA gene very similar to the trichloroethene reductive dehalogenase gene (tceA) of “Dehalococcoides ethenogenes” strain 195. A single strand conformation polymorphism (SSCP) method was developed allowing the simultaneous detection of the three rdhA genes and the estimation of their abundance. SSCP analysis of different SL2 cultures showed that one pceA gene was expressed during PCE dechlorination whereas the second was expressed during TCE dechlorination. The tceA gene was involved in cis-DCE dechlorination to ethene. Analysis of the internal transcribed spacer region between the 16S and 23S rRNA genes revealed two distinct sequences originating from Sulfurospirillum suggesting that two Sulfurospirillum populations were present in SL2. Whether each Sulfurospirillum population was catalyzing a different dechlorination step could however not be elucidated.     

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.     

Remediation of PCE-contaminated groundwater from an industrial site in southern Italy: A laboratory-scale study

Batch reactors and microcosms were used to evaluate groundwater bioremediation potential of tetrachloroethene (PCE) in the presence of additional pollutants present at a site located in the Apulia Region (SE Italy). Reductive dechlorination of PCE was studied under anaerobic conditions by comparing the effectiveness of three inocula: (a) soil sampled at the contaminated site, (b) anaerobic sludge from a municipal wastewater plant, and (c) an enriched dehalogenating culture containing Dehalococcoides species. In order to enhance dehalogenation, reactors inoculated with sludge were also amended with selected electron donors. Aerobic reactors were also established to study oxidative degradation of vinyl chloride (VC), that may accumulate after incomplete dechlorination of PCE.Results showed that consortia derived from anaerobic sludge and amended with electron donors quantitatively and incompletely degraded PCE to cis-dichloroethylene, whereas in reactors augmented with a dehalogenating culture complete dechlorination of PCE occurred even in the presence of additional toxic contaminants. The presence of Dehalococcoides spp. in the dehalogenating culture and its absence in reactors inoculated with anaerobic sludge was confirmed using FISH community analyses. In all cases, prolonged incubation periods were necessary for dechlorination. On the other hand, oxidative degradation of VC in aerobic reactors occurred after short lag times.     

Biochemical and molecular characterization of a tetrachloroethene dechlorinating Desulfitobacterium sp. strain Y51: a review

A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) at concentrations as high as 960 μM and as low as 0.06 μM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min⁻¹  mg protein⁻¹ . The apparent K _m values for PCE and TCE were 105.7 and 535.3 μM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE.

     

Enrichment and Characterization of a Trichloroethene-Dechlorinating Consortium Containing Multiple “Dehalococcoides” Strains

A microbial consortium that reductively dechlorinates trichloroethene, cis-1,2-dichloroethene (cis-DCE), and vinyl chloride (VC) to ethene with methanogenesis was enriched from chloroethene-contaminated soil from Japan. Dechlorination activity was maintained for over 4 years. Using quantitative polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis targeting the “Dehalococcoides” 16S rRNA gene, four strains were detected. Their growth and dechlorination activities were classified into two types: one that grows by converting cis-DCE to ethene and the other that grows by converting cis-DCE to VC. Then, the vcrA and bvcA genes encoding cis-DCE/VC reductive dehalogenases were detected. Inhibitors of methanogenesis (2-bromoethanesulfonate) and sulfidogenesis (molybdate) led to accumulation of cis-DCE and of VC respectively. These results suggest that methanogens and sulfate-reducing bacteria can play a significant role in dechlorination by “Dehalococcoides.”     

Benzoate-driven dehalogenation of chlorinated ethenes in microbial cultures from a contaminated aquifer

Microbial dehalogenation of tetrachloroethene (PCE) and cis-dichloroethene (cis-DCE) was studied in cultures from a continuous stirred tank reactor initially inoculated with aquifer material from a PCE-contaminated site. Cultures amended with hydrogen and acetate readily dechlorinated PCE and cis-DCE; however, this transformation was incomplete and resulted in the accumulation of chlorinated intermediates and only small amounts of ethene within 60 days of incubation. Conversely, microbial PCE and cis-DCE dechlorination in cultures with benzoate and acetate resulted in the complete transformation to ethene within 30 days. Community fingerprinting by denaturing gradient gel electrophoresis (DGGE) revealed the predominance of phylotypes closely affiliated with Desulfitobacterium, Dehalococcoides, and Syntrophus species. The Dehalococcoides culture VZ, obtained from small whitish colonies in cis-DCE dechlorinating agarose cultures, revealed an irregular cell diameter between 200 and 500 nm, and a spherical or biconcave disk-shaped morphology. These organisms were identified as responsible for the dechlorination of cis-DCE to ethene in the PCE-dechlorinating consortia, operating together with the Desulfitobacterium as PCE-to-cis-DCE dehalogenating bacterium and with a Syntrophus species as potential hydrogen-producing partner in cultures with benzoate. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.