A highly enriched culture that reductively dechlorinates trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC) to ethene without methanogenesis is described. The Dehalococcoides strain in this enrichment culture had a yield of (5.6 ؎ 1.4) ؋ 10 8 16S rRNA gene copies/mol of Cl ؊ when grown on VC and hydrogen. Unlike the other VC-degrading cultures described in the literature, strains VS and BAV1, this culture maintained the ability to grow on TCE with a yield of (3.6 ؎ 1.3) ؋ 10 8 16S rRNA gene copies/mol of Cl ؊ . The yields on an electron-equivalent basis measured for the culture grown on TCE and on VC were not significantly different, indicating that both substrates supported growth equally well. PCR followed by denaturing gradient gel electrophoresis, cloning, and phylogenetic analyses revealed that this culture contained one Dehalococcoides 16S rRNA gene sequence, designated KB-1/VC, that was identical (over 1,386 bp) to the sequences of previously described organisms FL2 and CBDB1. A second Dehalococcoides sequence found in separate KB-1 enrichment cultures maintained on cDCE, TCE, and tetrachloroethene was no longer present in the VC-H 2 enrichment culture. This second Dehalococcoides sequence was identical to that of BAV1. As neither FL2 nor CBDB1 can dechlorinate VC to ethene in a growth-related fashion, it is clear that current 16S rRNA gene-based analyses do not provide sufficient information to distinguish between metabolically diverse members of the Dehalococcoides group.Vinyl chloride (VC) is a known human carcinogen (ToxFAQs: vinyl chloride, Agency for Toxic Substances and Disease Registry, Centers for Disease Control and Prevention; http://www.atsdr.cdc.gov/toxfaq.html) and a common groundwater pollutant present at over 384 of 1,232 Final National Priority List sites identified by the U.S. Environmental Protection Agency (http://cfpub.epa.gov/supercpad/cursites /srchsites.cfm). It is the highest ranked organic compound on the 2001 Comprehensive Environmental Response, Compensation, and Liability Act Priority List of Hazardous Substances (http://www.atsdr.cdc.gov/01list.html). VC causes angiosarcoma, a rare form of liver cancer, as well as nervous system disorders and immune reactions. The U.S. Environmental Protection Agency has set a maximum contaminant level of 2 g/liter of drinking water for this chemical (http:// www.atsdr.cdc.gov/toxfaq.html). VC is found in the subsurface, due to incomplete degradation of other priority pollutants such as perchloroethene (PCE), trichloroethene (TCE), and 1,1,2-trichloroethane (11, 17; http://www.atsdr.cdc.gov /toxfaq.html). Other sources of VC in groundwater include waste from polyvinyl chloride plastic production. Although VC volatilizes rapidly and is subject to aerobic oxidation both abiotically and biotically (21), VC is persistent in the subsurface at many contaminated sites.Complete biological reductive dechlorination to ethene is gaining acceptance as a viable remediation method for some chlorinated ethene-contaminated sites (5...

A bench-scale study was performed to evaluate the enhancement of tetrachloroethene (PCE) dissolution from a dense nonaqueous phase liquid (DNAPL) source zone due to reductive dechlorination. The study was conducted in a pair of two-dimensional bench-scale aquifer systems using soil and groundwater from Dover Air Force Base, DE. After establishment of PCE source zones in each aquifer system, one was biostimulated (addition of electron donor) while the other was biostimulated and then bioaugmented with the KB1 dechlorinating culture. Biostimulation resulted in the growth of iron-reducing bacteria (Geobacter) in both systems as a result of the high iron content of the Dover soil. After prolonged electron donor addition methanogenesis dominated, but no dechlorination was observed. Following bioaugmentation of one system, dechlorination to ethene was achieved, coincident with growth of introduced Dehalococcoides and other microbes in the vicinity and downgradient of the PCE DNAPL (detected using DGGE and qPCR). Dechlorination was not detected in the nonbioaugmented system over the course of the study, indicating that the native microbial community, although containing a member of the Dehalococcoides group, was not able to dechlorinate PCE. Over 890 days, 65% of the initial emplaced PCE was removed in the bioaugmented, dechlorinating system, in comparison to 39% removal by dissolution from the nondechlorinating system. The maximum total ethenes concentration (3 mM) in the bioaugmented system occurred approximately 100 days after bioaugmentation, indicating that there was at least a 3-fold enhancement of PCE dissolution atthis time. Removal rates decreased substantially beyond this time, particularly during the last 200 days of the study, when the maximum concentrations of total ethenes were only about 0.5 mM. However, PCE removal rates in the dechlorinating system remained more than twice the removal rates of the nondechlorinating system. The reductions in removal rates over time are attributed to both a shrinking DNAPL source area, and reduced flow through the DNAPL source area due to bioclogging and pore blockage from methane gas generation.

Chlorinated solvents are among the most prevalent groundwater contaminants in the industrialized world. Biodegradation with Dehalococcoides-containing mixed cultures is an effective remediation technology. To elucidate transcribed genes in a Dehalococcoides-containing mixed culture, a shotgun metagenome microarray was created and used to investigate gene transcription during vinyl chloride (VC) dechlorination and during starvation (no chlorinated compounds) by a microbial enrichment culture called KB-1. In both treatment conditions, methanol was amended as an electron donor. Subsequently, spots were sequenced that contained the genes most differentially transcribed between the VC-degrading and methanol-only conditions, as well as spots with the highest intensities. Sequencing revealed that during VC degradation Dehalococcoides genes involved in transcription, translation, metabolic energy generation, and amino acid and lipid metabolism and transport were overrepresented in the transcripts compared to the average Dehalococcoides genome. KB-1 rdhA14 (vcrA) was the only reductive dehalogenase homologous (RDH) gene with higher transcript levels during VC degradation, while multiple RDH genes had higher transcript levels in the absence of VC. Numerous hypothetical genes from Dehalococcoides also had higher transcript levels in methanol-only treatments, indicating that many uncharacterized proteins are involved in cell maintenance in the absence of chlorinated substrates. In addition, microarray results prompted biological experiments confirming that electron acceptor limiting conditions activated a Dehalococcoides prophage. Transcripts from Spirochaetes, Chloroflexi, Geobacter, and methanogens demonstrate the importance of non-Dehalococcoides organisms to the culture, and sequencing of identified shotgun clones of interest provided information for follow-on targeted studies. C hlorinated solvents are among the most prevalent groundwater contaminants in industrialized countries, and their presence poses risks to human and environmental health. KB-1 is an anaerobic microbial community dominated by Dehalococcoides organisms that efficiently dechlorinates tetrachloroethene (PCE) and trichloroethene (TCE) to ethene (7,8,38). Dehalococcoides grows slowly in isolation and often has complex nutritional requirements, whereas Dehalococcoides growing in mixed communities grows and dechlorinates more rapidly, producing very robust cultures. This suggests beneficial relationships between nondechlorinating organisms and Dehalococcoides. It is therefore important to identify Dehalococcoides and non-Dehalococcoides genes within these communities that are expressed during dechlorination.There have been a few published microarray studies involving Dehalococcoides. In the first, Johnson et al. (14) examined transcription in Dehalococcoides strain 195 along a timeline from exponential to stationary phase in an effort to understand factors that limit the growth of this slow-growing isolate. They also used the arrays to investigate transc...

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