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.     

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.     

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.”     

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.     

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 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.     

Effect of pore velocity on biodegradation of <Emphasis Type="Italic">cis</Emphasis>-dichloroethene (DCE) in column experiments

Column experiments were conducted to evaluate the effect of pore velocity on the extent of biodegradation of cis-dichloroethene (cis-DCE) during transport in porous media. Columns were filled with homogeneous glass beads and inoculated with a culture capable of complete dechlorination of tetrachloroethene to ethene. A constant concentration of cis-DCE was maintained in the columns’ influent. Three different pore velocities were tested in duplicate, subjecting each column to a constant velocity. At high flow velocity, degradation of cis-DCE to ethene was nearly complete within the residence time of the columns. However, at medium and low flow velocities, incomplete dechlorination was observed. After 7 weeks, DNA was harvested from the columns to determine differences in the microbial populations. Results suggest that Dehalococcoides sp. were present in higher quantities in the high-velocity columns, consistent with the observed dechlorination. These results suggest that, at contaminated groundwater sites, heterogeneity of groundwater velocity may be one factor that contributes to heterogeneous distribution of biological activity.     

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.     

Biodegradation of cis-1,2-dichloroethylene and vinyl chloride in anaerobic cultures enriched from landfill leachate sediment under Fe(III)-reducing conditions

An anaerobic, Fe(III)-reducing enrichment culture, which originatedfrom a sediment sample collected at a landfill in Nanji-do, Seoul, Korea, was capable ofdegrading cis-1,2-dichloroethylene (cis-DCE) and vinylchloride (VC). Although it exhibited the ability under Fe(III)-reducing conditions, the chlorinated ethenes degradationwas not linked to the Fe(III) reduction. During cis-DCE degradation, no VC, ethene, or ethanewas detected through the experimental period. Also, this culture did not accumulate ethene andethane during the VC degradation. It was unlikely that cis-DCE was reductivelydechlorinated to VC and then the VC formed was dechlorinated fast enough. Because the kinetic datashowed that the rate of cis-DCE degradation was 3.5 times higher than that of VC. Whereasglucose supported the culture growth and the degradation, formate, acetate, butyrate, propionate,lactate, pyruvate, and yeast extract did not. The results appeared consistent with the involvement ofoxidative degradation mechanism rather than reductive dechlorination mechanism. The traits of the culturedescribed here are unusual in the anaerobic degradation of chlorinated ethenes and may be usefulfor searching an effective organism and mechanism regarding anaerobic cis-DCE and VC degradation.     

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.

     

Growth of Dehalobacter and Dehalococcoides spp. during Degradation of Chlorinated Ethanes

Mixed anaerobic microbial subcultures enriched from a multilayered aquifer at a former chlorinated solvent disposal facility in West Louisiana were examined to determine the organism(s) involved in the dechlorination of the toxic compounds 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA) to ethene. Sequences phylogenetically related to Dehalobacter and Dehalococcoides, two genera of anaerobic bacteria that are known to respire with chlorinated ethenes, were detected through cloning of bacterial 16S rRNA genes. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments after starvation and subsequent reamendment of culture with 1,2-DCA showed that the Dehalobacter sp. grew during the dichloroelimination of 1,2-DCA to ethene, implicating this organism in degradation of 1,2-DCA in these cultures. Species-specific real-time quantitative PCR was further used to monitor proliferation of Dehalobacter and Dehalococcoides during the degradation of chlorinated ethanes and showed that in fact both microorganisms grew simultaneously during the degradation of 1,2-DCA. Conversely, Dehalobacter grew during the dichloroelimination of 1,1,2-TCA to vinyl chloride (VC) but not during the subsequent reductive dechlorination of VC to ethene, whereas Dehalococcoides grew only during the reductive dechlorination of VC but not during the dichloroelimination of 1,1,2-TCA. This demonstrated that in mixed cultures containing multiple dechlorinating microorganisms, these organisms can have either competitive or complementary dechlorination activities, depending on the chloro-organic substrate.     

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.     

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.     

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.     

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.     

Development and characterization of DehaloR^2, a novel anaerobic microbial consortium performing rapid dechlorination of TCE to ethene

A novel anaerobic consortium, named DehaloR^2, that performs rapid and complete reductive dechlorination of trichloroethene (TCE) to ethene is described. DehaloR^2 was developed from estuarine sediment from the Back River of the Chesapeake Bay and has been stably maintained in the laboratory for over 2 years. Initial sediment microcosms showed incomplete reduction of TCE to DCE with a ratio of trans- to cis- isomers of 1.67. However, complete reduction to ethene was achieved within 10 days after transfer of the consortium to sediment-free media and was accompanied by a shift to cis-DCE as the prevailing intermediate metabolite. The microbial community shifted from dominance of the Proteobacterial phylum in the sediment to Firmicutes and Chloroflexi in DehaloR^2, containing the genera Acetobacterium, Clostridium, and the dechlorinators Dehalococcoides. Also present were Spirochaetes, possible acetogens, and Geobacter which encompass previously described dechlorinators. Rates of TCE to ethene reductive dechlorination reached 2.83 mM Cl⁻ d⁻¹ in batch bottles with a Dehalococcoides sp. density of 1.54E+11 gene copies per liter, comparing favorably to other enrichment cultures described in the literature and identifying DehaloR^2 as a promising consortium for use in bioremediation of chlorinated ethene-impacted environments.     

Dechlorination of three tetrachlorobenzene isomers by contaminated harbor sludge-derived enrichment cultures follows thermodynamically favorable reactions

Dechlorination patterns of three tetrachlorobenzene isomers, 1,2,3,4-, 1,2,3,5-, and 1,2,4,5-TeCB, were studied in anoxic microcosms derived from contaminated harbor sludge. The removal of doubly, singly, and un-flanked chlorine atoms was noted in 1,2,3,4- and 1,2,3,5-TeCB fed microcosms, whereas only singly flanked chlorine was removed in 1,2,4,5-TeCB microcosms. The thermodynamically more favorable reactions were selectively followed by the enriched cultures with di- and/or mono-chlorobenzene as the main end products of the reductive dechlorination of all three isomers. Based on quantitative PCR analysis targeting 16S rRNA genes of known organohalide-respiring bacteria, the growth of Dehalococcoides was found to be associated with the reductive dechlorination of all three isomers, while growth of Dehalobacter, another known TeCB dechlorinator, was only observed in one 1,2,3,5-TeCB enriched microcosm among biological triplicates. Numbers of Desulfitobacterium and Geobacter as facultative dechlorinators were rather stable suggesting that they were not (directly) involved in the observed TeCB dechlorination. Bacterial community profiling suggested bacteria belonging to the phylum Bacteroidetes and the order Clostridiales as well as sulfate-reducing members of the class Deltaproteobacteria as putative stimulating guilds that provide electron donor and/or organic cofactors to fastidious dechlorinators. Our results provide a better understanding of thermodynamically preferred TeCB dechlorinating pathways in harbor environments and microbial guilds enriched and active in anoxic TeCB dechlorinating microcosms.

     

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.     

Characterization of microbial community structure and population dynamics of tetrachloroethene-dechlorinating tidal mudflat communities

Tetrachloroethene (PCE) and trichloroethene (TCE) are common groundwater contaminants that also impact tidal flats, especially near urban and industrial areas. However, very little is known about dechlorinating microbial communities in tidal flats. Titanium pyrosequencing, 16S rRNA gene clone libraries, and dechlorinator-targeted quantitative real-time PCR (qPCR) characterized reductive dechlorinating activities and populations in tidal flat sediments collected from South Korea’s central west coast near Kangwha. In microcosms established with surface sediments, PCE dechlorination to TCE began within 10 days and 100% of the initial amount of PCE was converted to TCE after 37 days. cis-1,2-Dichloroethene (cis-DCE) was observed as dechlorination end product in microcosms containing sediments collected from deeper zones (i.e., 35–40 cm below ground surface). Pyrosequencing of bacterial 16S rRNA genes and 16S rRNA gene-targeted qPCR results revealed Desulfuromonas michiganensis-like populations predominanted in both TCE and cis-DCE producing microcosms. Other abundant groups included Desulfuromonas thiophila and Pelobacter acidigallici-like populations in the surface sediment microcosms, and Desulfovibrio dechloracetivorans and Fusibacter paucivorans-like populations in the deeper sediment microcosms. Dehalococcoides spp. populations were not detected in these sediments before and after incubation with PCE. The results suggest that tidal flats harbor novel, salt-tolerant dechlorinating populations and that titanium pyrosequencing provides more detailed insight into community structure dynamics of the dechlorinating microcosms than conventional 16S rRNA gene sequencing or fingerprinting methods.     

Isolation and characterization of tetrachloroethylene- and <Emphasis Type="Italic">cis</Emphasis>-1,2-dichloroethylene-dechlorinating propionibacteria

Two rapidly growing propionibacteria that could reductively dechlorinate tetrachloroethylene (PCE) and cis-1,2-dichloroethylene (cis-DCE) to ethylene were isolated from environmental sediments. Metabolic characterization and partial sequence analysis of their 16S rRNA genes showed that the new isolates, designated as strains Propionibacterium sp. HK-1 and Propionibacterium sp. HK-3, did not match any known PCE- or cis-DCE-degrading bacteria. Both strains dechlorinated relatively high concentrations of PCE (0.3 mM) and cis-DCE (0.52 mM) under anaerobic conditions without accumulating toxic intermediates during incubation. Cell-free extracts of both strains catalyzed PCE and cis-DCE dechlorination; degradation was accelerated by the addition of various electron donors. PCE dehalogenase from strain HK-1 was mediated by a corrinoid protein, since the dehalogenase was inactivated by propyl iodide only after reduction by titanium citrate. The amounts of chloride ions (0.094 and 0.103 mM) released after PCE (0.026 mM) and cis-DCE (0.05 mM) dehalogenation using the cell-free enzyme extracts of both strains, HK-1 and HK-3, were stoichiometrically similar (91 and 100%), indicating that PCE and cis-DCE were fully dechlorinated. Radiotracer studies with [1,2-¹⁴C] PCE and [1,2-¹⁴C] cis-DCE indicated that ethylene was the terminal product; partial conversion to ethylene was observed. Various chlorinated aliphatic compounds (PCE, trichloroethylene, cis-DCE, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,1-dichloroethane, 1,2-dichloroethane, 1,2-dichloropropane, 1,1,2-trichloroethane, and vinyl chloride) were degraded by cell-free extracts of strain HK-1.