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.

ABSTRACTThe groundwater contaminant 1,4-dioxane (dioxane) is transformed by several monooxygenase-expressing microorganisms, but only a few of these, includingPseudonocardia dioxanivoransstrain CB1190, can metabolize the compound as a sole carbon and energy source. However, nothing is yet known about the genetic basis of dioxane metabolism. In this study, we used a microarray to study differential expression of genes in strain CB1190 grown on dioxane, glycolate (a previously identified intermediate of dioxane degradation), or pyruvate. Of eight multicomponent monooxygenase gene clusters carried by the strain CB1190 genome, only the monooxygenase gene cluster located on plasmid pPSED02 was upregulated with dioxane relative to pyruvate. Plasmid-borne genes for putative aldehyde dehydrogenases, an aldehyde reductase, and an alcohol oxidoreductase were also induced during growth with dioxane. With both dioxane and glycolate, a chromosomal gene cluster encoding a putative glycolate oxidase was upregulated, as were chromosomal genes related to glyoxylate metabolism through the glyoxylate carboligase pathway. Glyoxylate carboligase activity in cell extracts from cells pregrown with dioxane and inRhodococcus jostiistrain RHA1 cells expressing the putative strain CB1190 glyoxylate carboligase gene further demonstrated the role of glyoxylate metabolism in the degradation of dioxane. Finally, we used13C-labeled dioxane amino acid isotopomer analysis to provide additional evidence that metabolites of dioxane enter central metabolism as three-carbon compounds, likely as phosphoglycerate. The routing of dioxane metabolites via the glyoxylate carboligase pathway helps to explain how dioxane is metabolized as a sole carbon and energy source for strain CB1190.

1,1,1-Trichloroethane (1,1,1-TCA) is a common groundwater contaminant that can be reductively dechlorinated to 1,1-dichloroethane (1,1-DCA) and monochloroethane, and can support the growth of certain dehalorespiring strains of Dehalobacter We used reductive dehalogenase cell-free extract assays (with reduced methyl viologen) and whole cell suspension dechlorination assays (with hydrogen) and a Dehalobacter-containing enrichment culture to explore the kinetics of l,1,1-TCA and 1,1-DCA reductive dechlorination in the presence of the common co-contaminants trichloroethene (TCE), cis-dichloroethene (cDCE), and vinyl chloride (VC). These chlorinated ethenes were most significant inhibitors of 1,1,1-TCA dechlorination in cell-free extracts, indicating direct effects on the reductive dehalogenase enzyme(s). The inhibition was present but less pronounced in whole cell suspension assays. None of the chlorinated ethenes inhibited 1,1-DCA dechlorination in cell-free extract assays, yet cDCE and particularly VC were inhibitors in whole cell assays, indicating an effect on Dehalobacter, but not on the dehalogenase enzyme(s). Marked differences in kinetic parameters for 1,1,1-TCA and 1,1-DCA, and an uncoupling of these two activities in cultures grown on 1,1-DCA compared to those grown on 1,1,1-TCA was strong evidence for the existence of distinct 1,1,1-TCA and 1,1-DCA reductive dehalogenase enzymes.

The bacterium Pseudonocardia dioxanivorans CB1190 grows on the cyclic ethers 1,4-dioxane (dioxane) and tetrahydrofuran (THF) as sole carbon and energy sources. Prior transcriptional studies indicated that an annotated THF monooxygenase (THF MO) gene cluster, thmADBC, located on a plasmid in CB1190 is upregulated during growth on dioxane. In this work, transcriptional analysis demonstrates that upregulation of thmADBC occurs during growth on the dioxane metabolite ␤-hydroxyethoxyacetic acid (HEAA) and on THF. Comparison of the transcriptomes of CB1190 grown on THF and succinate (an intermediate of THF degradation) permitted the identification of other genes involved in THF metabolism. Dioxane and THF oxidation activity of the THF MO was verified in Rhodococcus jostii RHA1 cells heterologously expressing the CB1190 thmADBC gene cluster. Interestingly, these thmADBC expression clones accumulated HEAA as a dead-end product of dioxane transformation, indicating that despite its genes being transcriptionally upregulated during growth on HEAA, the THF MO enzyme is not responsible for degradation of HEAA in CB1190. Similar activities were also observed in RHA1 cells heterologously expressing the thmADBC gene cluster from Pseudonocardia tetrahydrofuranoxydans K1.

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