Gasoline Oxygenate Biodegradation Processes in Mycobacterium vaccae JOB5

Abstract

Ether-based gasoline oxygenates are added to gasoline in the U.S. at concentrations ≤15% vol/vol to reduce carbon monoxide, hydrocarbon and particulate automobile emissions. These ether oxygenates, particularly methyl tertiary butyl ether (MTBE), have become a human health concern as they have been widely detected in drinking water sources. This study aimed to investigate the monooxygenase in Mycobacterium vaccae JOB5 previously found to cometabolize MTBE, focusing on the regulation and substrate range of this enzyme.

Our first study identified MTBE as an inducer of the monooxygenase enzyme in M. vaccae JOB5. In the presence of a nonalkane growth-supporting substrate, including organic acids, MTBE consumption was observed. The expected products of MTBE oxidation, tertiary butyl formate (TBF) and tertiary butyl alcohol (TBA) accumulated in the culture medium. Both the consumption of MTBE and the production of TBF and TBA were inhibited by acetylene, a specific inhibitor of alkane- and MTBE-oxidizing activity and by chloramphenical and rifampicin, transcriptional and translational inhibitors.

Further investigation into the regulation of the monooxygenase in M. vaccae JOB5 found that high concentrations of 1-propanol, a product of alkane oxidation by the monooxygenase, leads to inhibition of MTBE-oxidizing activity. The relationship between 1-propanol, MTBE, and the monooxygenase was not fully characterized in this study.

In addition to MTBE, ethyl tertiary butyl ether (ETBE) and tertiary amyl methyl ether (TAME) are alternate ether oxygenates added to gasoline. We found that propane-grown M. vaccae JOB5 cells are able to oxidize both ETBE and TAME and their alcohol products, TBA and tertiary amyl alcohol (TAA), respectively. Three lines of evidence suggest that the oxidation of these ethers and alcohols is initiated by the same monooxygenase responsible for MTBE-oxidation: the absence of a lag phase during incubation with propane-grown cells, inhibition of ether-oxidizing activity by acetylene, and the competitive interaction during incubation with propane.

Finally, we investigated the growth-supporting range of n-alkanes in the most well characterized alkane-oxidizing system, P. putida GPo1. This study led to the expansion of known n-alkanes which support growth in this organism to include the gaseous alkanes, n-butane and propane. The oxidation of these gaseous n-alkanes appears to be induced by the same alkane monooxygenase, AlkB, responsible for oxidation of the previously defined growth-supporting n-alkanes (C₅-C₁₂).

These studies have led to a better understanding of monooxygenase activity in both M. vaccae JOB5 and P. putida GPo1, including expanded knowledge of regulation and substrate ranges of this enzyme.