Petroleum contamination is ubiquitous worldwide, and poses significant health risks to humans, organisms, and the environment. Understanding the fate and behaviour of these chemicals is extremely important in order to predict and mitigate the effects of spills and accidental releases, and limit the exposure of these contaminants to humans and ecosystems. The physical and biological interactions with various petroleum hydrocarbons released into the environment were examined throughout this thesis in two different environmental settings; offshore bay sediments near Deepwater Horizon oil spill impacted sites, and an experimental aquifer injected with compounds representative of ethanol blended fuels. Stable carbon isotopes were used to identify carbon sources in a given environment as well as utilized by microbial communities during biodegradation of petroleum hydrocarbons. Patterns of n-alkanes, low levels of UCM and the lack of PAHs suggest hydrocarbons in Barataria Bay sediments were of dominantly terrestrial origin. Stable carbon isotope analysis of microbial lipids and n-alkanes indicate the presence of some petroleum residues, however there is no strong evidence of Deepwater Horizon oil. Dissolved ethanol, toluene, and MTBE were continuously injected into a pilot-scale laboratory tank simulating an unconfined sand aquifer contaminated with ethanol blended fuel. Ethanol, toluene and MTBE all experienced significant mass loss within the aquifer, which was attributed to biological degradation using stable carbon isotope analysis of residual hydrocarbons. Isotopic analysis of PLFA indicated a strong ethanol sourced signature used in microbial metabolism with some indications of an additional carbon sources such as toluene or MTBE. / Thesis / Master of Science (MSc)

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Aqua, a commercial product manufactured by BiOWiSH® Technologies, was utilized in this research to study its effectiveness to biodegrade methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA). Microcosms containing varying concentrations of MTBE and TBA as well as a growth media and mineral salt solution were examined. Analytical instrumentation used in this study included the use of a gas chromatograph-mass spectrometer (GC/MS) to determine concentrations of MTBE and TBA and a spectrophotometer to extrapolate approximate active biomass concentrations in each experiment. Four different environmental conditions were tested for both MTBE and TBA. The environmental conditions tested for each contaminant included: biodegradation under aerobic conditions, biodegradation under anaerobic conditions, biodegradation under denitrifying conditions, and biodegradation under aerobic conditions with glucose present. This study concluded that there is potential for degradation of MTBE and TBA using Aqua under the conditions tested. Maximum MTBE biodegradation was observed under aerobic conditions which yielded a first order rate constant of 0.019/hour and a 99.8 percent decrease in MTBE over 14 days. Maximum TBA biodegradation was observed under aerobic conditions with glucose present which yielded a first rate order constant of 0.009/hour and a 95.03 percent decrease in TBA concentrations over 14 days. It is presumed that under both conditions a monooxygenase enzymatic reaction involving Cytochrome P-450 aids in breaking down both MTBE and TBA. However, the results presented are indicative of biodegradation under lab conditions with little to no interference. Further research is needed to determine the effectiveness of Aqua utilizing groundwater or soil samples from MTBE or TBA contaminated sites in order to truly analyze Aqua’s potential to be used as a bioaugmentation product in real world applications.

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M.S.

The biochemical pathway for the anaerobic degradation of gallate, pyrogallol and phloroglucinol by Eubacterium oxidoreducens was investigated. Phloroglucinol reductase was purified 90-fold, from the soluble fraction of cell extract, to electrophoretic homogeneity. The enzyme was an α₂ homodimer with a native M_r of 78,000, did not contain metals or cofactors and was specific for phloroglucinol and NADPH with a K_m of 800 μM and 6.7 μM respectively at pH 6.8. The Km for phloroglucinol decreased with increasing pH. The enzyme catalyzed reaction was reversible and the equilibrium constant was 9.6. Dihydroresorcinol was a competitive inhibitor of the reverse reaction (K_i = 756 μM). Dihydrophloroglucinol produced in cell extract with H₂ as the reductant was identical to the compound produced by sodium borohydride reduction of phloroglucinol as shown by ¹H NMR spectroscopy. The ¹³C NMR spectrum was consistent with the structural assignment of dihydrophloroglucinol. The mechanism of the proposed enzymatically catalyzed reaction is proposed to involve transfer of a hydride equivalent from NADPH to the carbonyl carbon of the phloroglucinol dianion. Mutant strains of E. oxidoreducens that showed no gallate decarboxylase or dihydrophloroglucinol hydrolase activity were isolated after mutagenesis with ethylmethane sulfonate and emichment with ampicillin. The decarboxylase deficient mutants were unable to grow on gallate while pyrogallol and phloroglucinol supported growth. The hydrolase deficient mutants were unable to grow on any aromatic substrates and converted gallate to pyrogallol and dihydrophloroglucinol. The conversion of gallate to non-aromatic intermediates by cell extract of the wild-type stain was dependent on the presence of 1,2,3,5-benzenetetrol for the conversion of pyrogallol to phloroglucinol and on formate for the reduction of phloroglucinol to dihydrophloroglucinol. Transhydroxylase activity involved in the conversion of pyrogallol to phloroglucinol was induced by growth on aromatic substrates. The formate dehydrogenase was located in the soluble fraction of cell extract, and activity was protected from oxygen inactivation by sodium azide. The Km for formate and NADP was 290 μM and 140 μM respectively at pH 7.5. The pH optimum for activity was 7.5 and maximum activity was observed at a temperature of 50°C. / Ph. D.