Isolation and quantitative detection of tetrachloroethene (PCE)-dechlorinating bacteria in unsaturated subsurface soils contaminated with chloroethenes

The estimation of tetrachloethene (PCE) dechlorinating-activity and identification of PCE-dechlorinating bacteria were performed in 65 unsaturated subsurface soils (at a depth 30-60 cm) that were collected from 21 noncontaminated soils and 44 chloroethene-contaminated soils including four soils that dechlorinated PCE to 1,2-cis-dichloroethene (cisDCE) in situ. Sixteen out of the 44 PCE-contaminated soils and three out of the 21 noncontaminated soils dechlorinated PCE to trichloroethene and cisDCE but not vinyl chloride or ethene after a month of incubation with 0.1% yeast extract at 30 degrees C. Desulfitobacterium sp. strain B31e3 that can dechlorinate PCE to cisDCE was isolated from a soil that dechlorinated PCE to cisDCE in situ. 16S rRNA gene of this strain showed the closest similarity of 99.1% with that of Desulfitobacterium hafniense (formally frappieri) strain DP7. Real-time PCR using specific primer sets targeted to the 16S rRNA genes of the representative PCE-dechlorinating bacteria, Dehalococcoides spp., Desulfitobacterium spp., and Dehalobacter spp. were performed using five unsaturated subsurface soils that dechlorinated PCE and three that did not dechlorinate PCE. In two out of the five soils that dechlorinated PCE, Desulfitobacterium spp. (0.12, 0.38% of total bacteria) and Dehalobacter spp. (0.0045, 0.0061% of total bacteria) were detected, and in one of the five soils, only Desulfitobacterium spp. (0.042% of total bacteria) was detected. None of these representative PCE-dechlorinating bacteria were detected in two out of the five soils that dechlorinated PCE and in all of the three soils that did not dechlorinate PCE. Dehalococcoides spp. were not detected in any unsaturated subsurface soils used in this study. These results suggested the involvement of Desulfitobacterium spp. and probably Dehalobacter spp. rather than Dehalococcoides spp. in the dechlorination of PCE to cisDCE in unsaturated subsurface soils.