The purpose of this research was to investigate the microorganisms, genetics and biochemistry of anaerobic dechlorination of chlorinated ethanes, which are common groundwater contaminants. Specifically, this project used mixed microbial cultures to study the dechlorination of 1,2-dichloroethane (1,2-DCA), 1,1,2-trichloroethane (1,1,2-TCA) and 1,1,1-trichloroethane (1,1,1-TCA). A mixed microbial culture enriched from a contaminated multilayered aquifer in West Louisiana dechlorinated 1,2-DCA, 1,1,2-TCA, tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride (VC) to non-toxic ethene when amended with ethanol as the electron donor. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organisms Dehalobacter and Dehalococcoides spp. Denaturing gradient gel electrophoresis analysis and quantitative PCR (qPCR) with species-specific primers demonstrated that both organisms grew during the dichloroelimination of 1,2-DCA to ethene. Conversely, during the dichloroelimination of 1,1,2-TCA to VC only Dehalobacter grew, while during the reductive dechlorination of VC to ethene only Dehalococcoides grew. Further enrichment with 1,2-DCA, H2 and acetate yielded a co-culture of Dehalobacter and Acetobacterium spp. that did not dechlorinate other chlorinated ethanes or ethenes. Dehalobacter grew in the presence but not in the absence of 1,2-DCA, while Acetobacterium growth was not affected by 1,2-DCA. A novel putative Dehalobacter-associated 1,2-DCA reductive dehalogenase gene was identified and was shown to be transcribed only in the presence of 1,2-DCA. An enrichment microbial culture derived from a 1,1,1-TCA-contaminated site in the northeastern United States was also studied. This culture, referred to as MS, reductively dechlorinated 1,1,1-TCA to 1,1-dichloroethane (1,1-DCA) and then to monochloroethane (CA) when amended with methanol, ethanol, acetate and lactate. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organism Dehalobacter sp., whose growth during 1,1,1-TCA and 1,1-DCA dechlorination was confirmed by qPCR. In the presence of chlorinated ethenes, dechlorination 1,1,1-TCA by the culture MS was slowed, while dechlorination of 1,1-DCA was completely inhibited. Experiments with cell-free extracts and whole cell suspensions of culture MS suggested that chlorinated ethenes have direct inhibitory effects on 1,1,1-TCA reductive dehalogenase(s), while the inhibition of 1,1-DCA dechlorination may be due to effects on non-dehalogenase components of Dehalobacter sp. cells. Additionally, two novel reductive dehalogenase genes associated with 1,1,1-TCA reductive dechlorination were identified.

The purpose of this research was to investigate the microorganisms, genetics and biochemistry of anaerobic dechlorination of chlorinated ethanes, which are common groundwater contaminants. Specifically, this project used mixed microbial cultures to study the dechlorination of 1,2-dichloroethane (1,2-DCA), 1,1,2-trichloroethane (1,1,2-TCA) and 1,1,1-trichloroethane (1,1,1-TCA). A mixed microbial culture enriched from a contaminated multilayered aquifer in West Louisiana dechlorinated 1,2-DCA, 1,1,2-TCA, tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride (VC) to non-toxic ethene when amended with ethanol as the electron donor. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organisms Dehalobacter and Dehalococcoides spp. Denaturing gradient gel electrophoresis analysis and quantitative PCR (qPCR) with species-specific primers demonstrated that both organisms grew during the dichloroelimination of 1,2-DCA to ethene. Conversely, during the dichloroelimination of 1,1,2-TCA to VC only Dehalobacter grew, while during the reductive dechlorination of VC to ethene only Dehalococcoides grew. Further enrichment with 1,2-DCA, H2 and acetate yielded a co-culture of Dehalobacter and Acetobacterium spp. that did not dechlorinate other chlorinated ethanes or ethenes. Dehalobacter grew in the presence but not in the absence of 1,2-DCA, while Acetobacterium growth was not affected by 1,2-DCA. A novel putative Dehalobacter-associated 1,2-DCA reductive dehalogenase gene was identified and was shown to be transcribed only in the presence of 1,2-DCA. An enrichment microbial culture derived from a 1,1,1-TCA-contaminated site in the northeastern United States was also studied. This culture, referred to as MS, reductively dechlorinated 1,1,1-TCA to 1,1-dichloroethane (1,1-DCA) and then to monochloroethane (CA) when amended with methanol, ethanol, acetate and lactate. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organism Dehalobacter sp., whose growth during 1,1,1-TCA and 1,1-DCA dechlorination was confirmed by qPCR. In the presence of chlorinated ethenes, dechlorination 1,1,1-TCA by the culture MS was slowed, while dechlorination of 1,1-DCA was completely inhibited. Experiments with cell-free extracts and whole cell suspensions of culture MS suggested that chlorinated ethenes have direct inhibitory effects on 1,1,1-TCA reductive dehalogenase(s), while the inhibition of 1,1-DCA dechlorination may be due to effects on non-dehalogenase components of Dehalobacter sp. cells. Additionally, two novel reductive dehalogenase genes associated with 1,1,1-TCA reductive dechlorination were identified.

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