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...
The community composition of microbial cultures degrading tetrachloroethene (PCE), trichloroethene (TCE), cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC) to ethene was studied. A combination of PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and 16S rRNA gene sequence analysis revealed that all cultures contained Dehalococcoides populations, but that the populations of other organisms varied widely. Based on the sequences of cloned 16S rRNA genes, real-time PCR methods were developed for several of these phylotypes affiliated with the putative dechlorinators Sulfurospirillum and Geobacter, the putative methanogens Methanomethylovorans, Methanomicrobiales, Methanosaeta and Methanosarcina, the putative acetogens Acetobacterium, Spirochaetes, and Sporomusa, and the putative fermenters Bacteroidetes, Syntrophus, and Syntrophobacter. These novel quantitative PCR methods were then used to estimate relative abundances of each phylotype in several individual cultures maintained on each chlorinated ethene. Dehalococcoides populations were the dominant phylotypes assayed in most KB-1 cultures, agreeing with the DGGE and cloning results. A Geobacter phylotype was also strongly represented in most PCE and TCE cultures, but not in cDCE or VC cultures, suggesting a possible role for this organism as a PCE-to-cDCE dechlorinator. The Sulfurospirillum phylotype was estimated to comprise a minor fraction of 16S rRNA gene copies and did not appear to have an important role in dechlorination.
The population dynamics of a mixed microbial culture dechlorinating trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), 1,2-dichloroethane (1,2-DCA), and vinyl chloride (VC) to ethene were studied. Quantitative PCR revealed that Dehalococcoides, Geobacter, Sporomusa, Spirochaetes, and Methanomicrobiales phylotypes grew in short-term experiments. Both Geobacter and Dehalococcoides populations grew during TCE dechlorination to cDCE, but only Dehalococcoides populations grew during further dechlorination to ethene. The cell yields for Dehalococcoides determined in this study were similar on an electron equivalent basis regardless of the chlorinated compound transformed: (0.9+/-0.3) x 10(8)16S rRNA gene copies/microelectron equivalent (microeeq) ethene produced during cDCE dechlorination, (1.5 +/-0.3) x 10(8) copies/microeeq ethene produced during VC dechlorination, and (1.6+/-0.8) x 10(8) copies/ u,eeq ethene produced during 1,2-DCA dihaloelimination. The yield for the Geobacter population on TCE was estimated to be (1+/-0.5) x 10(8) copies/microeeq cDCE produced. Calculations showed that the Geobacter population was likely responsible for approximately 80% of the TCE dechlorinated to cDCE in this experiment. Acetogenesis by a Sporomusa population was the main competition to dechlorination for reducing equivalents. Sporomusa did not transform any chlorinated substrates tested, but was capable of converting methanol to acetate and hydrogen for dechlorination. Understanding the functions of various populations in mixed communities may explain why Dehalococcoides spp. are active at some sites and not others, and may also assist in optimizing the growth of bioaugmentation cultures, both in the laboratory and in the field.
Chloroform (CF), or trichloromethane, is an ubiquitous environmental pollutant because of its widespread industrial use, historically poor disposal and recalcitrance to biodegradation. Chloroform is a potent inhibitor of metabolism and no known organism uses it as a growth substrate. We discovered that CF was rapidly and sustainably dechlorinated in the course of investigating anaerobic reductive dechlorination of 1,1,1-trichloroethane in a Dehalobacter-containing culture. Like 1,1,1-trichloroethane dechlorination in this culture, CF dechlorination was a growth-linked respiratory process, requiring H(2) as an electron donor and CF as an electron acceptor. Moreover, the same specific reductive dehalogenase likely catalyzed both reactions. This Dehalobacter population appears specialized for substrates with three halogen substituents on the same carbon atom, with widespread implications for bioremediation.
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