Physiology and enzymology of aerobic MTBE and TBA biodegradation

Methyl tertiary butyl ether (MTBE), a widely used gasoline oxygenate, has been added to gasoline over the past 25 years to increase the octane rating and reduce air pollution from vehicle emissions. tertiary butyl alcohol (TBA) has also been used as a gasoline oxygenate and is a key intermediate in the biodegradation of MTBE. The frequent detection of these compounds as ground water contaminants has raised public health concerns and addresses the need for their remediation. This research focuses on the characterization of the physiology and enzymology of microorganisms able to grow on MTBE and TBA.;In this study, we isolated and characterized two aerobic bacterial strains able to utilize TBA as a sole source of carbon and energy. Strains, Hydrogenophaga G2B2 and Aquincola S1B1 rapidly oxidized TBA, but were unable to grow on MTBE or oxidize MTBE after growth on TBA. While strain G2B2 had a very limited growth substrate range, strain S1B1 grew on many postulated TBA oxidation intermediates including 2-methyl-1,2-propanediol (2M12PD), 2-hydroxyisobutyric acid (2HIBA), and hydroxyacetone. Using a novel approach combining a fluorinated analog of TBA, 2-trifluoromethyl-2-propanol (TFMP), with 19F-nuclear magnetic resonance (19F-NMR) spectroscopy, we also report the detection of the initial TBA biodegradation pathway intermediates 2M12PD and 2HIBA with both strains S1B1 and G2B2.;We also demonstrated the cometabolic oxidation of MTBE by a defined co-culture containing Pseudomonas mendocina KR1 and our isolate Aquincola S1B1. MTBE degradation was achieved through the n-alkane dependent cometabolic oxidation of MTBE by strain KR1 with the subsequent assimilation of TBA by strain S1B1. Further studies with strain S1B1 demonstrate that separate enzyme systems are required for TBA- and 2HIBA-oxidation and we report the identification of polypeptides induced after the growth of strain S1B1 on TBA. Our initial results suggest that these proteins are enzymes associated with TBA oxidation in this strain.;Our final study examined the biodegradation of MTBE by the MTBE-metabolizing strain Methylibium petroleiphilum PM1. Our results demonstrated that the MTBE biodegradation pathway of strain PM1 includes the intermediates TBA, 2M12PD, and 2HIBA. Furthermore, our results suggest that separate enzyme systems catalyze MTBE and TBA oxidation in this strain. We also identified a formaldehyde-activating protein in MTBE-grown cells, which may play a role in formaldehyde-detoxification during MTBE oxidation by strain PM1.;The research described in this dissertation contributes to the knowledge of MTBE and TBA biodegradation by strains able to utilize these compounds for growth. Our results suggest that there is considerable diversity between TBA metabolizing microorganisms and demonstrate a potential role of these microorganisms on the environmental fate of MTBE and TBA.