Chlorinated solvents have been widely used in various industrial process. The wide applications and improperly disposed has resulted in groundwater and soil contaminated with these compounds. In contaminated groundwater, trichloroethylene (TCE) and dichloroethylene (DCE) are two commonly detected volatile organic compounds (VOCs) and 1,2,3-trichloropropane (TCP) is a common co-contaminant. These three contaminants are known or suspended carcinogens. Maximum contaminant levels for TCE and DCE in drinking water has been set by EPA. In 2009, the U.S. EPA listed TCP in the Drinking Water Contaminant Candidate List 3(CCL3) (EPA 2009). To protect public health, these compounds need to be removed from contaminated aquifers and soils. While bioremediation technologies for chlorinated solvents have been successfully demonstrated in neutral pH aquifers, these technologies are often ineffective for remediating chlorinated solvents contamination in acidic aquifers (i.e., pH <5.5). Acidophilic methanotrophs have been detected in several low pH environments, but their presence and potential role in chlorinated solvents degradation in acidic aquifers is unknown. This study used stable isotope probing-based techniques to identify the presence and diversity of acidophilic methanotrophs in chlorinated solvent-contaminated acidic aquifers. By using TCE as a model groundwater contaminant, application of stable isotope probing-based technique has successfully identified active methanotrophs that were capable of degrading TCE in microcosms prepared from two low pH aquifer materials. A total of thirty-five clones of methanotrophs were derived from low pH microcosms in which methane and TCE degradation had been observed, with 29 clustered in ?-Proteobacteria and 6 clustered in ?-Proteobacteria. None of the clones has a high similarity to known acidophilic methanotrophs from other environments. The presence and diversity of particulate MMO and soluble MMO were also investigated. The pmoA gene was detected predominantly at one site, and the presence of a specific form of mmoX in numerous samples suggested that Methylocella spp. may be common in acidic aquifers. Finally, a methane-grown culture at pH 4 was enriched from an acidic aquifer and its ability to biodegrade various chlorinated ethenes was tested. Interestingly, the mixed culture rapidly degraded TCE and vinyl chloride, but not cis-1,2-DCE after growth on methane. Research efforts also extended to examine the degradative ability of pure acidophilic methanotrophs to chlorinated solvents, with respect to degradation kinetics and transformation capacity(Tc), and effects of carbon substrates on the degradative enzyme expression. Two acidophilic methanotrophic strains were used as model microorganisms. Both strains were able to grow on multi-carbon sources, while only methane-grown cells showed non-specific enzyme activity based on naphthalene oxidation tests. Positive PCR results confirmed that Methylocella tundrae carried the cluster gene of propane monooxygenase and probably could use propane as the carbon source. While the strain grew well on isopropanol (an immediate product of propane oxidation), the strain grew poorly with propane. Interestingly, the isopropanol-grown cells showed negative results in naphthalene oxidation assay, suggesting that isopropanol might not be an inducer for propane monooxygenase. Methane-grown Methylocella tundrae and Methylocystis bryophila were able to degrade TCE and cis-1,2-DCE. Only Methylocella tundrae could degrade TCP. The results of this study suggest that aerobic biodegradation of TCE and other chlorinated solvents in low pH groundwater may be facilitated by methanotrophic bacteria, and that there are potentially a wide variety of different strains that inhabit acidic aquifers.
