Characterizing MTBE Cometabolism and Propane Metabolism by Mycobacterium austroafricanum JOB5

Abstract

Characterizing MTBE Cometabolism and Propane Metabolism by Mycobacterium austroafricanum JOB5. (Under the direction of Michael R. Hyman.) Cometabolic transformations are unable to support cell growth. This process is often catalyzed by, and superimposed upon, enzyme systems expressed to catalyze carbon- or energy-yielding reactions. Biodegradation of the gasoline additive methyl tertiary butyl ether (MTBE) is known to be superimposed upon a propane-oxidizing system in the aerobic bacterium Mycobacterium austroafricanum (vaccae) JOB5. Taking a whole-cell approach, we investigated the physiology of propane metabolism and MTBE cometabolism in this strain.

Multiple major gasoline components are frequent co-contaminants with MTBE in the environment, and we determined the impacts of these hydrocarbons on the cometabolism of both MTBE and its commonly encountered metabolite, tertiary butyl alcohol (TBA). Most of the hydrocarbons tested supported cell growth and concurrent MTBE and TBA oxidation occurred without affecting final culture optical density. Results suggest hydrocarbon-grown cells simultaneously expressed more than one alkane-oxidizing enzyme system.

Nuclear magnetic resonance spectroscopy (NMR) was used to study the pathway of MTBE oxidation in propane-grown cells of strain JOB5. We confirmed the existence of predicted intermediates, including a hemiacetal, formate and formaldehyde. Hydroxyisobutyraldehyde, a predicted intermediate in MTBE oxidation by some bacterial strains was not detected, despite attempts to promote its accumulation. As the pathway of MTBE oxidation progressed, the rate of daughter product oxidation decreased, which may be preventing MTBE-dependent cell growth in strain JOB5.

Propane metabolism was examined using a series of growth experiments and substrate oxidation assays. We observed the simultaneous production, and later consumption of both 1- and 2-propanol during cell growth. This divergent oxidation of propane was apparently followed by the divergent oxidation of propionate and the divergent oxidation of acetone. Our results suggest at least two CO2-fixation steps are involved in propane metabolism in strain JOB5.

Finally, we used NMR to contribute to several studies that characterized the pathway of (i) MTBE oxidation by Nitrosomonas europaea or (ii) bacterial oxidation of a fluorinated analog of TBA. The later study identified a compound that may serve as a tracer for TBA degradation in situ.