An anaerobic microbial consortium (referred to as ANAS) that reductively dechlorinates trichloroethene (TCE) completely to ethene with the transient production of cisdichloroethene (cDCE) and vinyl chloride was enriched from contaminated soil obtained from Alameda Naval Air Station. ANAS uses lactate as its electron donor and has been functionally stable for over 2 years. Following a brief exposure to oxygen, a subculture (designated VCC) derived from ANAS could dechlorinate TCE only to vinyl chloride with lactate as its electron donor. Three molecular methods were used concurrently to characterize the community structure of ANAS and VCC: clone library construction/clone sequencing, terminal restriction fragment length polymorphism (T-RFLP) analysis, and fluorescent in situ hybridization (FISH) with rRNA probes. The community structure of ANAS did not change significantly over the course of a single feeding/dechlorination cycle, and only minor fluctuations occurred over many feeding cycles spanning the course of 1 year. Clone libraries and T-RFLP analyses suggested that ANAS was dominated by populations belonging to three phylogenetic groups: Dehalococcoides species, Desulfovibrio species, and members of the Clostridiaceae (within the low G + C Gram-positives). FISH results suggest that members of the Cytophaga/Flavobacterium/Bacteroides (CFB) cluster and high G + C Gram-positives (HGCs) were numerically important in ANAS despite their under-representation in the clone libraries. Parallel analyses of VCC samples suggested that Dehalococcoides species and Clostridiaceae were only minor populations in this community. Instead, VCC had increased populations of organisms in the beta and gamma subclasses of the Proteobacteria as well as significant populations of organisms in the CFB cluster. It is possible that symbiotic interactions are occurring between some of ANAS's phylogenetic groups under the enrichment conditions, including interspecies hydrogen transfer from Desulfovibrio species to Dehalococcoides species. However, the nucleic acid-based analyses performed here would need to be supplemented with chemical species data in order to test any hypotheses about functional roles of various community members. Additionally, these results suggest that an organism outside the Dehalococcoides genus may be capable of dechlorinating cDCE to vinyl chloride.

Time-series stable carbon isotope monitoring of volatile organic compounds (VOCs) atthe Idaho National Engineering and Environmental Laboratory's (INEEL) field site Test Area North (TAN) was conducted during a pilot study to investigate the treatment potential of using lactate to stimulate in situ biologic reductive dechlorination of trichloroethene (TCE). The isotope ratios of TCE and its biodegradation byproducts, cis-dichloroethene (c-DCE), trans-dichloroethene (t-DCE), vinyl chloride (VC), and ethene, in groundwater samples collected during the pilot studywere preconcentrated with a combination of purge-and-trap and cryogenic techniques in order to allow for reproducible isotopic measurements of the low concentrations of these compounds in the samples (down to 0.04 microM, or 5 ppb, of TCE). Compound-specific stable isotope monitoring of chlorinated solvents clearly differentiated between the effects of groundwater transport, dissolution of DNAPL at the source, and enhanced bioremediation. Isotope data from all wells within the zone of lactate influence exhibited large kinetic isotope effects during the reduction of c-DCE to VC and VC to ethene. Despite these large effects, the carbon isotope ratio of ethene in all these wells reached the carbon isotope ratios of the initial dissolved TCE, confirming the complete conversion of dissolved TCEto ethene. Conversely, the carbon isotope ratios of t-DCE were only marginally affected during the study, indicating that minimal biologic degradation of t-DCE was occurring.

Stable isotope analysis is recognized as a powerful tool for monitoring, assessing, and validating in-situ bioremediation processes. In this study, kinetic carbon isotope fractionation factors (epsilon) associated with the aerobic biodegradation of vinyl chloride (VC), cis-1,2-dichloroethylene (cDCE), and trichloroethylene (TCE) were examined. Of the three solvents, the largest fractionation effects were observed for biodegradation of VC. Both metabolic and cometabolic VC degradation were studied using Mycobacterium aurum L1 (grown on VC), Methylosinus trichosporium OB3b (grown on methane), Mycobacterium vaccae JOB5 (grown on propane), and two VC enrichment cultures seeded from contaminated soils of Alameda Point and Travis Air Force Base, CA. M. aurum L1 caused the greatest fractionation (epsilon = -5.7) while for the cometabolic cultures, epsilon values ranged from -3.2 to -4.8. VC fractionation patterns for the enrichment cultures were within the range of those observed for the metabolic and cometabolic cultures (epsilon = -4.5 to -5.5). The fractionation for cometabolic degradation of TCE by Me. trichosporium OB3b was low (epsilon = -1.1), while no quantifiable carbon isotopic fractionation was observed during the cometabolic degradation of cDCE. For all three of the tested chlorinated ethenes, isotopic fractionation measured during aerobic degradation was significantly smaller than that reported for anaerobic reductive dechlorination. This study suggests that analysis of compound-specific isotopic fractionation could assist in determining whether aerobic or anaerobic degradation of VC and cDCE predominates in field applications of in-situ bioremediation. In contrast, isotopic fractionation effects associated with metabolic and cometabolic reactions are not sufficiently dissimilar to distinguish these processes in the field.