The use of enhanced in situ anaerobic bioremediation (EISB) and bioaugmentation in fractured bedrock is limited compared to its use in granular media. We evaluated EISB for the treatment of trichloroethene (TCE)-impacted groundwater in fractured carbonate rock at a site in Southern Ontario, Canada, with cool average groundwater temperature (∼ 13 °C). Borehole-connectivity, contaminant concentrations, and groundwater properties were investigated. Changes in dechlorinating and nondechlorinating populations (fermenters, acetogens, methanogens, and sulfate reducers) were assessed via quantitative PCR (qPCR). During biostimulation with ethanol, concentrations of TCE daughter products cis-dichloroethene (cDCE) and vinyl chloride (VC) decreased in association with an enrichment of vcrA (VC reductive dehalogenase)-carrying Dehalococcoides, whereas ethene production was only moderate. Following bioaugmentation with the mixed dechlorinating culture KB-1, greater concentrations of chloride-a product of dechlorination-was observed in most wells; in addition, ethene production increased significantly in monitoring well locations that had strong hydraulic connectivity to the groundwater recirculation system, while Dehalococcoides and vcrA concentrations did not appreciably vary. Interestingly, increases of 3-4 orders of magnitude of an ethanol-fermenting Bacteroidetes population also present in KB-1 were correlated to improved conversion to ethene, an observation which suggests there could be a causal relationship-for example, better syntrophy and/or synergy among bacterial populations.

bThe WBC-2 consortium is an organohalide-respiring anaerobic microbial enrichment culture capable of dechlorinating 1,1,2,2-tetrachloroethane (TeCA) to ethene. In the WBC-2 culture, TeCA is first transformed to trans-dichloroethene (tDCE) by dichloroelimination; tDCE is subsequently transformed to vinyl chloride (VC) and then to ethene by hydrogenolysis. Analysis of 16S rRNA gene clone libraries from culture DNA revealed sequences from three putative dechlorinating organisms belonging to Dehalococcoides, Dehalobacter, and Dehalogenimonas genera. Quantitative PCR primers were designed for each of these sequences, and their abundance was quantified in enrichment cultures over time. These data revealed that complete dechlorination of TeCA to ethene involves all three organisms. Dehalobacter spp. grew during the dihaloelimination of TeCA to tDCE, while Dehalococcoides and Dehalogenimonas spp. grew during hydrogenolysis of tDCE to ethene. This is the first time a genus other than Dehalococcoides has been implicated in dechlorination of tDCE to VC. 1,1,2,2-Tetrachloroethane (TeCA) was one of the first chlorinated solvents produced in North America before World War I (4). Chronic exposure to TeCA can cause liver damage and possibly cancer (29). Today, TeCA is no longer manufactured yet remains present at historically contaminated sites. TeCA has been detected at 326 of 1,699 sites recommended for the National Priorities List (28) and is ranked 147th out of 275 chemicals on the 2007 CERCLA hazardous chemicals list (1). In anaerobic groundwater, TeCA degrades abiotically by dehydrohalogenation to trichloroethene (TCE) and biotically by reductive dechlorination to the nontoxic end product ethene (19). The transformation products produced during microbially mediated TeCA dechlorination, particularly vinyl chloride (VC), are more toxic than the parent compound (6); therefore, the ability to rapidly degrade these daughter products is essential for effective bioremediation. Several microbial consortia and isolated bacterial strains capable of dechlorinating TeCA have been described previously (2-4, 19, 22, 25, 26, 27, 30). Pure strains degrade TeCA incompletely: Desulfuromonas michiganensis (26) and Desulfitobacterium hafniense strain Y51 (27) are reported to dechlorinate TeCA to cis-dichloroethene (cDCE) and Dehalogenimonas lykanthroporepellans (strains BL-DC-8 and BL-DC-9) is reported to dechlorinate TeCA to a mixture of cDCE and trans-dichloroethene (tDCE) (22,26,27). To date, none of the characterized strains of Dehalogenimonas are capable of dechlorinating chlorinated ethenes, including the DCE isomers and VC (22,26,27).The mixed microbial culture WBC-2, enriched from sediment from the West Branch Canal Creek (20), dechlorinates TeCA completely and stoichiometrically to ethene. The WBC-2 enrichment culture has been shown to degrade a wide range of contaminants, including TeCA, 1,1,2-trichloroethane (1,1,2-TCA), 1,2-dichloroethane (1,2-DCA), TCE, cDCE, tDCE, and VC (16,19) and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) ...