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) x 10(8) 16S rRNA gene copies/micromol 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) x 10(8) 16S rRNA gene copies/micromol 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.

FIG. 1.

VC dechlorination in 20% (vol/vol) transfer cultures of KB-1/VC-H₂. Note that 20 μmol of VC/bottle corresponded to 190 μM (aqueous) VC. Error bars are standard deviations of results for three replicate cultures.

FIG.2.

The KB-1/VC-H₂ culture, DAPI stained and viewed at an original magnification of ×1,000.

FIG. 3.

(A) PCR-DGGE targeting Dehalococcoides in four KB-1 enrichment cultures. (B) General bacterial PCR-DGGE, illustrating that the KB-1/VC-H₂ culture (left lane) had lost all but the Dehalococcoides band compared to the mixed KB-1 culture (right lane).

FIG. 4.

Increase in Dehalococcoides 16S rRNA gene copy number during VC and TCE degradation. Solid diamonds correspond to VC degradation, and open diamonds correspond to TCE degradation. The x axis error bars represent the 10% uncertainty in gas chromatography measurements. The y axis error bars represent the error in quantitative PCR measurements, estimated to be 10%, based on analysis of replicate samples.