Substrate oxidation by methanotrophs expressing particulate methane monooxygenase (pMMO): A study of whole-cell oxidation of trichloroethylene and its potential use for environmental remediation

Many studies have examined rapid trichloroethylene (TCE) co-metabolism by methanotrophs expressing soluble methane monooxygenase (sMMO). Most known methanotrophs, however, can not express sMMO and rely on particulate methane monooxygenase (pMMO). Furthermore, as sMMO is only expressed at low copper/biomass ratios, pMMO may predominate in the environment. Therefore, the goal of this research is to provide more information on TCE degradation by methanotrophs expressing pMMO to better understand methanotrophic-mediated TCE bioremediation.; Addition of copper enhanced TCE oxidation by Methylosinus trichosporium OB3b expressing pMMO but did not dramatically affect Methylomicrobium album BG8. As M. album BG8 can express only pMMO, it may have a better copper uptake system than M. trichosporium OB3b which can express both forms of MMO. Addition of formate as an external source of reducing equivalents increased TCE oxidation by both strains. Therefore, these results suggest to provide high copper and formate concentrations to enhance TCE bioremediation by methanotrophs expressing pMMO.; The products of TCE oxidation by in vivo pMMO of M. album BG8 were determined using radiotracer techniques. TCE was suspected to be oxidized into TCE-epoxide which was spontaneously hydrolyzed to form glyoxylate as the primary aqueous product. Glyoxylate was subsequently oxidized by pMMO to form formate and CO2. Formate could also be further oxidized to CO2 by formate dehydrogenase in whole-cells. Unlike sMMO-mediated TCE oxidation, chloral and dichloroacetate were not detected.; In experiments of simultaneous oxidation of methane and chlorinated hydrocarbons, TCE and dichloromethane (DCM) did not significantly affect methane oxidation by M. trichosporium OB3b expressing pMMO in the absence of formate. If formate was added, TCE acted as a non-competitive inhibitor while DCM acted as a competitive inhibitor of methane oxidation by M. trichosporium OB3b expressing pMMO. Therefore, a simple competitive inhibition model can not be applied to accurately predict the time required in achieving the goal of TCE bioremediation by methanotrophs expressing pMMO.; As in vivo pMMO and sMMO activities show different characteristics, an assay to distinguish them is needed to optimize bioremediation. Phenylacetylene inhibited in vivo sMMO activity at 100 muM and was shown to be a mechanism-based inactivator of sMMO. In vivo pMMO activity was affected at a similar level only in the presence of 1000 muM phenylacetylene. These studies provide the foundation for using phenylacetylene in environmental microbiology.