A laboratory microcosm study and a pilot scale field test were conducted to evaluate biostimulation and bioaugmentation to dechlorinate tetrachloroethene (PCE) to ethene at Kelly Air Force Base. The site groundwater contained about 1 mg/L of PCE and lower amounts of trichloroethene (TCE) and cis-1,2-dichloroethene (cDCE). Laboratory microcosms inoculated with soil and groundwater from the site exhibited partial dechlorination of TCE to cDCE when amended with lactate or methanol. Following the addition of a dechlorinating enrichment culture, KB-1, the chlorinated ethenes in the microcosms were completely converted to ethene. The KB-1 culture is a natural dechlorinating microbial consortium that contains phylogenetic relatives of Dehalococcoides ethenogenes. The ability of KB-1 to stimulate biodegradation of chlorinated ethenes in situ was explored using a closed loop recirculation cell with a pore volume of approximately 64,000 L The pilot test area (PTA) groundwater was first amended with methanol and acetate to establish reducing conditions. Under these conditions, dechlorination of PCE to cDCE was observed. Thirteen liters of the KB-1 culture were then injected into the subsurface. Within 200 days, the concentrations of PCE, TCE, and cis-1,2-DCE within the PTA were all below 5 microg/L, and ethene production accounted for the observed mass loss. The maximum rates of dechlorination estimated from field date were rapid (half-lives of a few hours). Throughout the pilot test period, groundwater samples were assayed for the presence of Dehalococcoides using both a Dehalococcoides-specific PCR assay and 16S rDNA sequence information. The sequences detected in the PTA after bioaugmentation were specific to the Dehalococcoides species in the KB-1 culture. These sequences were observed to progressively increase in abundance and spread downgradient within the PTA. These results confirm that organisms in the KB-1 culture populated the PTA aquifer and contributed to the stimulation of dechlorination beyond cDCE to ethene.

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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.

Stable carbon isotope fractionation is a valuable tool for monitoring natural attenuation and to establish the fate of groundwater contaminants. In this study, we measured carbon isotope fractionation during aerobic and anaerobic degradation of two chlorinated benzenes: monochlorobenzene (MCB) and 1,2,4-trichlorobenzene (1,2,4-TCB). MCB isotope fractionation was measured in anaerobic methanogenic microcosms, while 1,2,4-TCB isotope experiments were carried out in both aerobic and anaerobic microcosms. Large isotope fractionation was observed in both the anaerobic microcosm experiments. Enrichment factors (ε) for anaerobic reductive dechlorination of MCB and 1,2,4-TCB were -5.0‰ ± 0.2‰ and -3.0‰ ± 0.4‰, respectively. In contrast, no significant isotope fractionation was found during aerobic microbial degradation of 1,2,4-TCB. The cleavage of a C-Cl σ bond occurs during anaerobic reductive dechlorination of MCB and 1,2,4-TCB, while no σ bond cleavage is involved during aerobic degradation via dioxygenase. The difference in isotope fractionation for aerobic versus anaerobic biodegradation of MCB and 1,2,4-TCB can be explained by the difference in the initial step of aerobic versus anaerobic biodegradation pathways.

Reliance on bioremediation to remove benzene from anoxic environments has proven risky for decades but for unknown reasons. Research has revealed a strong link between anaerobic benzene biodegradation and the enrichment of highly specific microbes, including Thermincola in the family Peptococcaceae and the deltaproteobacterial Candidate Sva0485 clade. Using aquifer materials from Canadian Forces Base Borden, we compared five bioremediation approaches in batch microcosms. Under conditions simulating natural attenuation or sulfate biostimulation, benzene was not degraded after 1–2 years of incubation and no enrichment of known benzene-degrading microbes occurred. In contrast, nitrate-amended microcosms reported benzene biodegradation coincident with significant growth of Thermincola spp., along with a functional gene presumed to catalyze anaerobic benzene carboxylation (abcA). Inoculation with 2.5% of a methanogenic benzene-degrading consortium containing Sva0485 (Deltaproteobacteria ORM2) resulted in benzene biodegradation in the presence of sulfate or under methanogenic conditions. The presence of other hydrocarbon co-contaminants decreased the rates of benzene degradation by a factor of 2 to 4. Tracking the abundance of the abcA gene and 16S rRNA genes specific for benzene-degrading Thermincola and Sva0485 is recommended to monitor benzene bioremediation in anoxic groundwater systems to further uncover growth-rate-limiting conditions for these two intriguing phylotypes.