Regulation and Environmental Abundance of Methyl-tert-Butyl Ether Degradation Genes of Methylibium petroleiphilum PM1

Methyl-tert-butyl ether (MTBE) and its degradation by-product tertiary butyl alcohol (TBA) are widespread contaminants detected frequently in groundwater in California. They are recalcitrant to biodegradation under anaerobic conditions. MTBE was used as a fuel-oxygenate for almost two decades, an important source of this contamination being leaking underground fuel storage tanks. Such leaks result in the presence of other gasoline components as groundwater co-pollutants, such as BTEX (benzene, toluene, ethylbenzene and xylenes) along with MTBE. Several bacteria are known to aerobically degrade MTBE. Of these, Methylibium petroleiphilum strain PM1 can serve as a model organism to understand the molecular mechanisms of regulation of the MTBE degradation pathway, since its genome has been sequenced. This dissertation aimed to understand these mechanisms, and relate them with MTBEdegradation in the environment.;We profiled the changes in transcription of MTBE-degradation genes in strain PM1 after exposure to MTBE, benzene and ethylbenzene. We demonstrated the direct mRNA-based evidence of high induction of genes mdpA (MTBE monooxygenase), mdpJ (TBA hydroxylase) and bmoA (benzene monooxygenase) in response to MTBE, TBA and benzene, respectively. Transcription of mdpA and mdpJ decreased 4-fold and 2.5-fold, respectively, in the presence of benzene, which also corresponded to their slower degradation rates. Our results indicate that ethylbenzene, identified previously as an inhibitor of MTBE degradation in some bacteria, directly inhibits transcription of mdpA, mdpJ and bmoA in strain PM1. Further, we demonstrated the involvement of a putative transcriptional activator gene, mdpC, in the induction of genes mdpA and mdpJ of strain PM1 by creating an mdpC- mutant strain. Our results indicate that an additional independent mechanism may be involved in the induction of mdpJ in the presence of TBA. We then examined the potential role of these MTBE- and TBA-responsive genes as biomarkers for MTBE-degradation in contaminated environments. The abundance of these genes in DNA extracted from groundwater sampled at sites that were treated via air-sparging, and at untreated sites that were undergoing monitored natural attenuation, were analyzed. We found strong correlation between PM1 genes, and MTBE and TBA concentrations at several untreated sites. Conversely, all sites undergoing treatment did not show a relationship between contaminant concentrations and degradation genes. In conclusion, a deeper understanding of regulatory mechanisms and genes involved in MTBE biodegradation, and correlation of gene abundance with contaminant concentration despite variation in geographical and hydrogeological characteristics among various environmental sites, indicate a potential for using functional-gene abundances as biomarkers for active degradation processes in the field.;Organic and inorganic contaminants pose threat to water quality globally. In the developing world, pathogenic bacterial contamination is another major factor impacting the quality of drinking water. We conducted community-engaged research with a local non-profit partner to evaluate the installation, continued use and efficiency of coliform removal by bio-sand filters in Nkokonjeru, Uganda. We found that regular and timely monitoring of installed filters, and relevant re-training of personnel involved in construction, installation and maintenance of the filters was essential for their sustained efficient use. Additionally, colloidal silver coated ceramic bricks were also evaluated for coliform removal in harvested rainwater based on standard coliform tests as well as the amount of silver released in solution after repeated use. We observed complete disinfection of harvested rainwater amended with canine feces within 24 h -- 96 h in 200 L tanks. Silver was maintained in a steady-state concentration of ∼60 ppb for up to 60 days in tanks with repeated use. Therefore, our results indicated that silver-coated ceramic bricks can serve as a feasible approach for small-scale water treatment, although their adaptation requires further research regarding socio-economic acceptance of this product in communities. -.