Assessing Anaerobic Bio-Oxidation of Vinyl Chloride and Ethene

Perchloroethene (PCE) and trichloroethene (TCE) are two of the most commonly used chlorinated solvents around the world. These compounds and their dechlorination daughter products have become the most prevalent organic contaminants found in a majority of hazardous waste sites in the United States. Since implementation of traditional remediation methods would cost hundreds of billions of dollars, there has been tremendous interest in bioremediation as a low cost alternative to achieving remedial goals. Anaerobic reductive dechlorination is the most prevalent form of bioremediation in most locations. The ultimate daughter products from reductive dechlorination of PCE and TCE are ethene and ethane, which are non-hazardous. However, at many hazardous waste sites, the sum of the daughter products often does not account for the amount of PCE and TCE consumed. At least two explanations have been offered for this phenomenon. First, downgradient sampling of plumes may not be accurately representing where the plume is. Second, it is possible that the lesser chlorinated products, in particular vinyl chloride (VC) and ethene, could be undergoing anaerobic or aerobic oxidation. If this occurs, CO2 and Cl- are the major daughter products. While both are nonhazardous, it is far more difficult to show that these compounds were formed from VC and ethene, rather than other compounds. An alternative pathway is bio-oxidation. In this case, the chlorinated ethenes and/or ethene are used as an electron donor and are oxidized to CO2, biomass, and Cl- (except with ethene). For the chlorinated ethenes, only cis-1,2-dichloroethene (cDCE) and VC have been shown to undergo anaerobic oxidation; there are no reports of anaerobic oxidation of PCE or TCE. Bio-oxidation of ethene has been well documented for aerobic conditions, while there is only one report of ethene bio-oxidation under sulfate-reducing conditions. Furthermore, almost nothing is known about the microbes responsible for anaerobic oxidation of cDCE, VC and ethene. The overall objective of this research was to culture and ultimately isolate and characterize microbes that are capable of using VC or ethene as a sole source of carbon and energy by anaerobic oxidation. To accomplish this objective, four types of cultures were used: 1) Microcosms that were started in a previous project were continued as part of this thesis research, with the objective of determining the fate of [14C]VC and [14C]ethene; 2) One of the nitrate-amended microcosms that was previously developed exhibited presumptive anaerobic biodegradation of VC; it was used as an inoculum for preparation of transfer cultures, with the objective of developing a VC-oxidizing enrichment culture under nitrate-reducing conditions; 3) A new set of microcosms was prepared with sediment and groundwater from a wetland at the Savannah River Site in which a plume of TCE discharges and undergoes reductive dechlorination to ethene and ethane. This is the same location that was used to develop microcosms that reduced ethene to ethane, a subset of which was sent to the University of Toronto, where evidence was obtained for transformation of ethene to methane. The objective for this research was to replicate the results for ethene transformation to methane; and 4) A sulfate-reducing enrichment culture developed by Fullerton and Zinder at Cornell University was used to evaluate the fate of 14C-ethene, with the objective of determining the extent of mineralization and/or transformation to organic acids. The experiments performed during this research with the Cornell culture unequivocally demonstrated that it mineralizes ethene under anaerobic conditions, with nearly 90% of the 14C-ethene recovered as 14CO2. This is the first culture developed with the ability to anaerobically grow on ethene as its sole carbon and energy source. The Cornell culture provides an opportunity to make a significant breakthrough in developing methods to evaluate the extent of in situ anaerobic oxidation of ethene. Bio-oxidation of VC was observed in a number of microcosms, with activity sustained for the longest period of time in a treatment amended with nitrate. Bio-oxidation also occurred in an enrichment culture inoculated with one of the microcosms. However, the results are considered equivocal, due to uncertainty over the potential role of oxygen contamination, most likely via diffusion through the serum bottle septa. Several lines of evidence support the hypothesis of oxygen contamination, including bio-oxidation of methane (an uncommon anaerobic process) along with VC, and oxidation of nitrite to nitrate (not known to occur under anaerobic conditions) in several of the enrichment bottles. On the other hand, oxygen contamination seems improbable, considering that the bottles were incubated in an anaerobic chamber over the interval when VC biodegradation occurred. Also, the rate of VC biodegradation was often much faster than would be expected if oxygen was diffusing into the bottles at a slow rate. If anaerobic bio-oxidation did occur, it remains uncertain what the electron acceptor was (or it fermentation occurred), since bio-oxidation occurred in enrichment bottles that were not amended with nitrate or nitrite, as well as in ones that were amended with nitrate. Additional research is needed to conclusively determine the role of oxygen in the observed oxidation of VC. Over this course of this thesis research and prior studies on the same topic by students at Clemson University, hundreds of microcosms were prepared with soil and groundwater from ten sites, and usually multiple locations from these sites, to evaluate the potential for anaerobic oxidation of VC and ethene. The most common results, confirmed in this study, were either no activity or reduction occurred (i.e., VC to ethene, and ethene to ethane). A few of the bottles showed signs of anaerobic oxidation, but conclusive evidence for this process has not yet been obtained, with the exception of the Cornell ethene oxidation culture. Numerous variations in microcosm preparation were attempted, but none resulted in reliable oxidative activity. Taken together, these results suggest, to the extent that anaerobic oxidation does occur in situ, it is not an especially common process. An alternative explanation is that the process is common, but the approaches used to replicate it in the laboratory have been inadequate