Characterization of a Highly Enriched Dehalococcoides -Containing Culture That Grows on Vinyl Chloride and Trichloroethene

Abstract

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...

Guided cobalamin biosynthesis supports Dehalococcoides mccartyi reductive dechlorination activity

“…The Dehalococcoides-containing consortium KB-1 used for bioaugmentation contains methanogens and rstb.royalsocietypublishing.org Phil Trans R Soc B 368: 20120320 CO 2 -reducing acetogens [29]. Both methanogenic Archaea and acetogenic Bacteria are known to synthesize cobamides with different lower ligands (figure 1).…”

Quantifying Enhanced Microbial Dehalogenation Impacting the Fate and Transport of Organohalide Mixtures in Contaminated Sediments

“…are widespread in the environment (Dennis et al, 2003;Duhamel et al, 2004;Fennell et al, 2001;Gu et al, 2004;Kassenga et al, 2004;Löffler et al, 2000;Major et al, 2002;Richardson et al, 2002;Smits et al, 2004) and have been shown to dechlorinate PCDD/Fs (Bunge et al, 2001(Bunge et al, , 2003Yoshida et al, 2005;Hiraishi et al, 2005;Ahn et al, 2007. Our previous study demonstrated the increase of a Dehalococcoides population along with the dechlorination of weathered PCBs in Anacostia River (Washington, DC) sediment microcosms, suggesting their potential role in PCB dechlorination .…”

“…2-2 and 2-4), however, could not explain the differences in the PCB dechlorination activity observed in the two different haloprimer treatments. The 16S rRNA genes of Chloroflexi species are highly conserved and do not correspond with different dechlorination substrate specificities Duhamel et al, 2004;He et al, 2005;Kube et al, 2005;Sung et al, 2006). For this reason, Band 1 (Fig.…”

“…A single-nucleotide primer extension assay is available also for the three subgroups in group II Dehalococcoides species involved in anaerobic dechlorination processes (Nikolausz et al, 2008;Yohda et al, 2008). However, a limitation of these 16S rRNA gene based assays is that Dehalococcoides species typically represent a small fraction of the total bacterial community (Major et al, 2002;Lendvay et al, 2003;Amos et al, 2008) and their 16S rRNA genes are highly conserved Duhamel et al, 2004;He et al, 2005;Kube et al, 2005;Sung et al, 2006). As a result, it is difficult to detect closely related strains within the dechlorinating community by monitoring changes in 16S rRNA phylotypes alone.…”

“…Sample DNA and 10-fold serially diluted 16S rRNA gene standards were amplified in parallel on an iQ5 real-time PCR system (Bio-Rad Laboratories, Hercules, CA). Each 25 µL of reaction contained 1 µL of sample or standard DNA, 1X iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA), and 0.2 µM (each) Dehalococcoidesspecific 16S rRNA gene primers (1F, 5'-GATGAACGCTAGCGGCG-3' and 259R, 5'-CAGACCAGCTACCGATCGAA-3') (Duhamel et al, 2004; The temperature profile was: 5 min at 95 °C; 40 cycles of 30 sec at 95 °C; 30 sec at 55 °C; and 30 sec at 72 °C. Each analysis was followed by melt curve analysis which confirmed that only Dehalococcoides specific sequences (same single melting point as the cloned standard) were amplified.…”