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.
-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 TCEdegrading 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 chloroethenedegrading 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.
Mixed anaerobic microbial subcultures enriched from a multilayered aquifer at a former chlorinated solvent disposal facility in West Louisiana were examined to determine the organism(s) involved in the dechlorination of the toxic compounds 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA) to ethene. Sequences phylogenetically related to Dehalobacter and Dehalococcoides, two genera of anaerobic bacteria that are known to respire with chlorinated ethenes, were detected through cloning of bacterial 16S rRNA genes. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments after starvation and subsequent reamendment of culture with 1,2-DCA showed that the Dehalobacter sp. grew during the dichloroelimination of 1,2-DCA to ethene, implicating this organism in degradation of 1,2-DCA in these cultures. Species-specific real-time quantitative PCR was further used to monitor proliferation of Dehalobacter and Dehalococcoides during the degradation of chlorinated ethanes and showed that in fact both microorganisms grew simultaneously during the degradation of 1,2-DCA. Conversely, Dehalobacter grew during the dichloroelimination of 1,1,2-TCA to vinyl chloride (VC) but not during the subsequent reductive dechlorination of VC to ethene, whereas Dehalococcoides grew only during the reductive dechlorination of VC but not during the dichloroelimination of 1,1,2-TCA. This demonstrated that in mixed cultures containing multiple dechlorinating microorganisms, these organisms can have either competitive or complementary dechlorination activities, depending on the chloro-organic substrate.
Dehalobacter and "Dehalococcoides" spp. were previously shown to be involved in the biotransformation of 1,1,2-trichloroethane (1,1,2-TCA) and 1,2-dichloroethane (1,2-DCA) to ethene in a mixed anaerobic enrichment culture. Here we report the further enrichment and characterization of a Dehalobacter sp. from this mixed culture in coculture with an Acetobacterium sp. Through a series of serial transfers and dilutions with acetate, H 2 , and 1,2-DCA, a stable coculture of Acetobacterium and Dehalobacter spp. was obtained, where Dehalobacter grew during dechlorination. The isolated Acetobacterium strain did not dechlorinate 1,2-DCA. Quantitative PCR with specific primers showed that Dehalobacter cells did not grow in the absence of a chlorinated electron acceptor and that the growth yield with 1,2-DCA was 6.9 (؎0.7) ؋ 10 7 16S rRNA gene copies/mol 1,2-DCA degraded. PCR with degenerate primers targeting reductive dehalogenase genes detected three distinct Dehalobacter/Desulfitobacterium-type sequences in the mixed-parent culture, but only one of these was present in the 1,2-DCA-H 2 coculture. Reverse transcriptase PCR revealed the transcription of this dehalogenase gene specifically during the dechlorination of 1,2-DCA. The 1,2-DCA-H 2 coculture could dechlorinate 1,2-DCA but not 1,1,2-TCA, nor could it dechlorinate chlorinated ethenes. As a collective, the genus Dehalobacter has been show to dechlorinate many diverse compounds, but individual species seem to each have a narrow substrate range.
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