Biodegradation of Trichloroethylene and Dichloromethane in Contaminated Soil and Groundwater

Soil column and serum bottle microcosm experiments were conducted to investigate the potential for in situ anaerobic bioremediation of trichloroethy lene (TCE) and dichloromethane (DCM) at the Pinellas site near Largo, Florida. Soil columns with continuous groundwater recycle were used to evaluate treatment with complex nutrients (casamino acids, methanol, lactate, sulfate); benzoate and sulfate; and methanol. The complex nutrients drove microbial dechlorination of TCE to ethene, whereas the benzoate/sulfate and methanol supported microbial dechlorination of TCE only to cis-1 ,2-dichloroethylene (cDCE). Microbial sulfate depletion in the benzoate/sulfate column allowed further dechlorination of cDCE to vinyl chloride. Serum bottle microcosms were used to investigate TCE dechlorination and DCM biodegradation in Pinellas soil slurries bioaugmented with liquid from the soil columns possessing TCE-dechlorinating activity and DCM biodegradation by indigenous microorganisms. Bioaugmented soil microcosms showed immediate TCE dechlorination in the microcosms with methanol or complex nutrients, but no dechlorination in the benzoate/sulfate microcosm. DCM biodegradation by indigenous microorganisms occurred in soil microcosms amended with either benzoate/sulfate or methanol, but not with complex nutrients. Bioaugmentation stimulated DCM biodegradation in both complex nutrient and methanol-amended microcosms, but appeared to inhibit DCM biodegradation in benzoate/sulfate-amended microcosms. TCE dechlorination occurred before DCM biodegradation in bioaugmented microcosms when both compounds were present.     

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.     

Dynamics of reductive TCE dechlorination in two distinct H<Subscript>2</Subscript> supply scenarios and at various temperatures

Anaerobic microbial dechlorination of trichloroethene (TCE) by a mixed, Dehalococcoides containing culture was investigated at different temperatures (4–60 °C) using propionate and lactate as a slow- and fast-releasing hydrogen (H₂) source, respectively. Distinct temperature-dependent dynamics of substrate fermentation and H₂ levels could explain observed patterns of dechlorination. While varying the temperature caused changes in rate, the overall pattern of dechlorination was characteristic of the supplied electron donor. Feeding cultures with a rapidly fermentable substrate such as lactate generally resulted in high H₂ concentrations and fast and complete dechlorination accompanied by rapid methanogenesis. In contrast, low H₂ release rates resulting from fermentation of propionate were associated with 2 to 3−fold longer time frames necessary for complete dechlorination at intermediate temperatures (15–30 °C). A lag-phase prior to dechlorination of cis-dichloroethene (cDCE), together with a characteristic build-up of H₂ and methane, was consistently observed at slow H₂ supply. At temperatures of 10 °C and lower, the system remained in this lag phase and no dechlorination past cDCE was observed within the experimental time frame. However, when lactate was the substrate, complete dechlorination of TCE occurred within 74 days at 10 °C, accompanied by methane production. The choice of fermentable substrate decisively influenced the rate and degree of dechlorination at an electron donor/TCE ratio as high as 666:1. Temperature-dependent H₂ levels resulting from fermentation of different substrates could be satisfactorily explained through thermodynamic calculations of the Gibbs free energy yield assuming a constant metabolic energy threshold of −20 kJ/(mol reaction).     

Microbial reductive dechlorination of trichloroethene to ethene with electrodes serving as electron donors without the external addition of redox mediators

In situ bioremediation of industrial chlorinated solvents, such as trichloroethene (TCE), is typically accomplished by providing an organic electron donor to naturally occurring dechlorinating populations. In the present study, we show that TCE dechlorinating bacteria can access the electrons required for TCE dechlorination directly from a negatively polarized (?450 mV vs. SHE) carbon paper electrode. In replicated batch experiments, a mixed dechlorinating culture, also containing Dehalococcoides spp., dechlorinated TCE to cis‐dichloroethene (cis‐DCE) and lower amounts of vinyl chloride (VC) and ethene using the polarized electrode as the sole electron donor. Conversely, neither VC nor ethene formation occurred when a pure culture of the electro‐active microorganism Geobacter lovleyi was used, under identical experimental conditions. Cyclic voltammetry tests, carried out on the filter‐sterilized supernatant of the mixed culture revealed the presence of a self‐produced redox mediator, exhibiting a midpoint potential of around ?400 mV (vs. SHE). This yet unidentified redox‐active molecule appeared to be involved in the extracellular electron transfer from the electrode to the dechlorinating bacteria. The ability of dechlorinating bacteria to use electrodes as electron donors opens new perspectives for the development of clean, versatile, and efficient bioremediation systems based on a controlled subsurface delivery of electrons in support of biodegradative metabolisms and provides further evidence on the possibility of using conductive materials to manipulate and control a range of microbial bioprocesses. Biotechnol. Bioeng. 2009;103: 85–91. © 2008 Wiley Periodicals, Inc.     

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.     

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.     

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.     

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.     

Bioremediation of Hexachlorocyclohexane Isomers,Chlorinated Benzenes,and Chlorinated Ethenes in Soil and Fractured Dolomite

Groundwater at an industrial site is contaminated with α hexachlorocyclohexane (HCH) and γ -HCH (i.e., lindane) (0.3 to 0.5 ppm). Other contaminants in the 1 to 15 ppm range include 1,2,4-trichlorobenezene (TCB), 1,2-dichlorobenzene (DCB), 1,3-DCB, 1,4-DCB, chlorobenzene (CB), benzene, trichloroethene (TCE), and cis-1,2-dichloroethene (cDCE). The aquifer consists of a shallow layer of soil over fractured dolomite, where most of the contaminant mass resides. The objective of this study was to compare (1) anaerobic reductive dechlorination of the polychlorinated contaminants, followed by aerobic biodegradation of the daughter products (mainly DCBs, CB, and benzene); and (2) aerobic biodegradation of α - and γ -HCH, TCB, DCBs, CB, and benzene, followed by anaerobic reduction of TCE and cDCE to ethene. Conventional wisdom suggests that sequential anaerobic and aerobic conditions are desirable for bioremediating sites contaminated by mixtures of polychlorinated organics. The results of this microcosm study suggest that a sequential aerobic and anaerobic approach may be more successful, although implementing this in the field presents some major challenges. In the dolomite microcosms incubated under aerobic conditions first (59 days), α - and γ -HCH were biodegraded close to the maximum contaminant level for lindane; all of the aromatic compounds were consumed; and there was partial removal of TCE and cDCE (presumptively via cometabolism). The subsequent switch to anaerobic conditions (day 101) yielded reductive dechlorination of the remaining TCE; a significant level of ethene was produced, although some cDCE and VC persisted. In contrast, sequential anaerobic (393 days) and aerobic treatment (498 days) for the dolomite microcosms was ineffective in completely removing the aromatic compounds, α -HCH, cDCE, and VC. For the soil microcosms, both treatment sequences were effective, most likely reflecting a greater abundance of the necessary microbes and electron donor in this part of the site.     

Molinate biodegradation in soils: natural attenuation versus bioaugmentation

The aims of the present study were to assess the potential of natural attenuation or bioaugmentation to reduce soil molinate contamination in paddy field soils and the impact of these bioremediation strategies on the composition of soil indigenous microbiota. A molinate mineralizing culture (mixed culture DC) was used as inoculum in the bioaugmentation assays. Significantly higher removal of molinate was observed in bioaugmentation than in natural attenuation microcosms (63 and 39 %, respectively) after 42 days of incubation at 22 °C. In the bioaugmentation assays, the impact of Gulosibacter molinativorax ON4^T on molinate depletion was observed since the gene encoding the enzyme responsible for the initial molinate breakdown (harboured by that actinobacterium) was only detected in inoculated microcosms. Nevertheless, the exogenous mixed culture DC did not overgrow as the heterotrophic counts of the bioaugmentation microcosms were not significantly different from those of natural attenuation and controls. Moreover, the actinobacterial clone libraries generated from the bioaugmentation microcosms did not include any 16S rRNA gene sequences with significant similarity to that of G. molinativorax ON4^T. The multivariate analysis of the 16S rRNA DGGE patterns of the soil microcosm suggested that the activity of mixed culture DC did not affect the soil bacterial community structure since the DGGE patterns of the bioaugmentation microcosms clustered with those of natural attenuation and controls. Although both bioremediation approaches removed molinate without indigenous microbiota perturbation, the results suggested that bioaugmentation with mixed culture DC was more effective to treat soils contaminated with molinate.     

Bioaugmentation of butane-utilizing microorganisms to promote cometabolism of 1,1,1-trichloroethane in groundwater microcosms

The transformation of 1,1,1-trichloroethane (1,1,1-TCA) in ioaugmented and non-augmented microcosms was evaluated. The microcosms contained roundwater and aquifer materials from a test site at Moffett Field, Sunnyvale, CA. The initial inoculum for bioaugmentation was a butane-utilizing enrichment from the subsurface of the Hanford DOE site. The non-augmented microcosm required 80 days of incubation before butane-utilization was observed while the augmented microcosms required 3 days. Initially the augmented microcosms were effective in transforming 1,1,1-TCA, but their transformation ability decreased after prolonged incubation. The non-augmented microcosms initially showed limited 1,1,1-TCA transformation but improved with time. After 440 days, both the non-augmented and augmented microcosms had similar transformation yields (0.04 mg 1,1,1-TCA/mg butane) and had similar microbial composition (DNA fingerprints). Subsequent microcosms, when bioaugmented with a Hanford enrichment that was repeatedly grown in 100% mineral media, did not effectively grow or transform 1,1,1-TCA under groundwater nutrient conditions. Microcosm tests to study the effect of mineral media on transformation ability were performed with the Hanford enrichment. Microcosms with 50% mineral media in groundwater most effectively utilized butane and transformed 1,1,1-TCA, while microcosms with groundwater only and microcosms with 5% mineral media in groundwater lost their 1,1,1-TCA transformation ability. DNA fingerprinting indicated shifts in the microbial composition with the different mineral media combinations. Successful bioaugmentation was achieved by enriching butane-utilizers from Moffett Field microcosms that were effective in groundwater with no mineral media added. The results suggest that successful in-situ bioaugmentation might be achieved through the addition of enriched cultures that perform well under subsurface nutrient conditions.     

A 1,1,1-trichloroethane-degrading anaerobic mixed microbial culture enhances biotransformation of mixtures of chlorinated ethenes and ethanes

1,1,1-trichloroethane (1,1,1-TCA) is a common groundwater pollutant as a result of improper disposal and accidental spills. It is often found as a cocontaminant with trichloroethene (TCE) and inhibits some TCE-degrading microorganisms. 1,1,1-TCA removal is therefore required for effective bioremediation of sites contaminated with mixed chlorinated organics. This study characterized MS, a 1,1,1-TCA-degrading, anaerobic, mixed microbial culture derived from a 1,1,1-TCA-contaminated site in the northeastern United States. MS reductively dechlorinated 1,1,1-TCA to 1,1-dichloroethane (1,1-DCA) and then to monochloroethane (CA) but not further. Cloning of bacterial 16S rRNA genes revealed among other organisms the presence of a Dehalobacter sp. and a Desulfovibrio sp., which are both phylogenetically related to known dehalorespiring strains. Monitoring of these populations with species-specific quantitative PCR during degradation of 1,1,1-TCA and 1,1-DCA showed that Dehalobacter proliferated during dechlorination. Dehalobacter growth was dechlorination dependent, whereas Desulfovibrio growth was dechlorination independent. Experiments were also performed to test whether MS could enhance TCE degradation in the presence of inhibiting levels of 1,1,1-TCA. Dechlorination of cis-dichloroethene (cDCE) and vinyl chloride (VC) in KB-1, a chloroethene-degrading culture used for bioaugmentation, was inhibited with 1,1,1-TCA present. When KB-1 and MS were coinoculated, degradation of cDCE and VC to ethene proceeded as soon as the 1,1,1-TCA was dechlorinated to 1,1-DCA by MS. This demonstrated the potential application of the MS and KB-1 cultures for cobioaugmentation of sites cocontaminated with 1,1,1-TCA and TCE.     

The role of microbial reductive dechlorination of TCE at a phytoremediation site

In April 1996, a phytoremediation field demonstration site at the Naval Air Station, Fort Worth, Texas, was developed to remediate shallow oxic ground water (< 3.7 m deep) contaminated with chlorinated ethenes. Microbial populations were sampled in February and June 1998. The populations under the newly planted cottonwood trees had not yet matured to an anaerobic community that could dechlorinate trichloroethene (TCE) to cis-1,2-dichloroethene (DCE); however, the microbial population under a mature (approximately 22-year-old) cottonwood tree about 30 m southwest of the plantings had a mature anaerobic population capable of dechlorinating TCE to DCE, and DCE to vinyl chloride (VC). Oxygen-free sediment incubations with contaminated groundwater also demonstrated that resident microorganisms were capable of the dechlorination of TCE to DCE. This suggests that a sufficient amount of organic material is present for microbial dechlorination in aquifer microniches where dissolved O2 concentrations are low. Phenol, benzoic acid, acetic acid, and a cyclic hydrocarbon, compounds consistent with the degradation of root exudates and complex aromatic compounds, were identified by gas chromatography/mass spectrometry (GC/MS) in sediment samples under the mature cottonwood tree. Elsewhere at the site, transpiration and degradation by the cottonwood trees appears to be responsible for loss of chlorinated ethenes.     

Biodegradation of Polycyclic Aromatic Hydrocarbons (PAHs) by Trichoderma reesei FS10-C and Effect of Bioaugmentation on an Aged PAH-Contaminated Soil

ABSTRACT?The co-metabolism of benzo[a]pyrene (B[a]P) and the capacity of the fungus Trichoderma reesei FS10-C to bioremediate an aged polycyclic aromatic hydrocarbon (PAH)-contaminated soil were investigated. The fungal isolate removed about 54% of B[a]P (20 mg L^?1) after 12 days of incubation with glucose (10 g L^?1) supplementation as a co-metabolic substrate. Bioaugmented microcosms showed a 25% decrease in total PAH concentrations in soil after 28 days, and the degradation percentages of 3-, 4-, and 5(+6)-ring PAHs were 36%, 35%, and 25%, respectively. In addition, bioaugmented microcosms exhibited higher dehydrogenase (DHA) and fluorescein diacetate hydrolysis (FDAH) activities and increased average well-color development (AWCD), Shannon-Weaver index (H), and Simpson index (D) significantly. Principal component analysis (PCA) also distinguished clear differentiation between treatments, indicating that bioaugmentation restored the microbiological function of the PAH-contaminated soil. The results suggest that bioaugmentation by T. reesei FS10-C might be a promising bioremediation strategy for aged PAH-contaminated soils.     

Evaluation of Whey for Bioremediation of Trichloroethene Source Zones

The effectiveness of whey as an electron donor that stimulates bioremediation and enhances dissolution of trichloroethene (TCE) dense non-aqueous phase liquid (DNAPL) was investigated. Laboratory experiments were conducted to evaluate increased mass transfer of TCE from the DNAPL to the aqueous phase in abiotic batch microcosms amended with several concentrations of whey, and in abiotic columns using high- and low-concentration whey mixtures. The effective solubility of TCE was a factor of 6 higher in microcosms amended with 10% w/w whey compared to 1% w/w whey or nanopure water. Increased aqueous-phase concentrations of TCE were a function of both the concentration of whey and time. In the columns, a factor of 5 increase in TCE concentrations was observed in the effluent during amendment with 10% w/w whey compared to potable water and 1% w/w whey. A field study involving three whey injections was performed at a site that had been actively undergoing bioremediation in a residual source area using lactate for 5 years. Results of the field test show a factor of 3 increase in total molar concentrations of chloroethenes and ethene following injection of 10% w/w whey compared to 5% lactate. In addition, complete dechlorination of TCE to ethene continued.     

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.     

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 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.