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This study used polyacrylamide gel electrophoresis to study the acid-phenol soluble proteins of five strains (A. vinelandii 12837, A. vinelandii 0, A. chroococcum 8004, A. macrocytogenes 8702, A. tumefaciens) of bacteria grown on Burk's nitrogen-free media, Trypticase Soy Broth, and 0.3% butanol medium. The results showed that the protein patterns can be used for the identification and possibly the taxonomic classification of the Azotobacter. The change of phenotype of the bacteria in different media followed the change of protein quantity and quality. There was no absolute similarity between any two of the species studied and this suggests a genetically heterogenous group of organisms while the amount of common proteins suggests close genetic relationships. Further studies are necessary to confirm the status of A. tumefaciens.

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Sphingopyxis (formerly Sphingomonas) alaskensis, a numerically abundant species isolated from Alaskan waters and the North Sea represents one of the only pure cultures of a typical oligotrophic ultramicrobacterium isolated from the marine environment. In this study, physiological and molecular characterization of an extinction dilution isolate from the North Pacific indicate that it is a strain of Sphingopyxis alaskenis, extending the known geographical distribution of this strain and affirming its importance as a model marine oligotroph. Given the importance of open ocean systems in climatic processes, it is clearly important to understand the physiology and underlying molecular biology of abundant species, such as S. alaskensis, and to define their role in biogeochemical processes. S. alaskensis is thought to proliferate by growing slowly on limited concentrations of substrates thereby avoiding outright starvation. In order to mimic environmental conditions chemostat culture was used to study the physiology of this model oligotroph in response to slow growth and nutrient limitation. It was found that the extent of nutrient limitation and starvation has fundamentally different consequences for the physiology of oligotrophic ultramicrobacteria compared with well-studied copiotrophic bacteria (Vibrio angustum S14 and Escherichia coli). For example, growth rate played a critical role in hydrogen peroxide resistance of S. alaskensis with slowly growing cells being 10, 000 times more resistant than fast growing cells. In contrast, the responses of V. angustum and E. coli to nutrient availability differed in that starved cells were more resistant than growing cells, regardless of growth rate. In order to examine molecular basis of the response to general nutrient limitation, starvation and oxidative stress in S. alaskensis we used proteomics to define differences in protein profiles of chemostat-grown cultures at various levels of nutrient limitation. High-resolution two-dimensional electrophoresis (2DE) methods were developed and 2DE protein maps were used to define proteins regulated by the level of nutrient limitation. A number of these proteins were identified with the aid of mass spectrometry and cross-species database matching. The identified proteins are involved in fundamental cellular processes including protein synthesis, protein folding, energy generation and electron transport, providing an important step in discovering the molecular basis of oligotrophy in this model organism.

<p> Biodegradation of benzene, a common groundwater contaminant, occurs readily in the presence of oxygen; however, at contaminated sites, aerobic bacteria often deplete the available oxygen, resulting in anaerobic conditions. Field and laboratory studies have shown that the anaerobic biodegradation of other aromatic hydrocarbons such as toluene occurs readily, while anaerobic benzene biodegradation has only been documented in a handful of studies. Despite these difficulties, benzene biodegradation has been shown to occur under iron-reducing, sulphate-reducing and methanogenic conditions, but not under nitrate-reducing conditions.</p> <p> The goal of this thesis research was to enrich and characterize the benzene-degrading microbial populations in microcosms and transfer cultures derived from soil from four different sites. Cultures were amended with potential exogenous electron acceptors (nitrate, sulphate, ferric iron) and the rates of biodegradation under different terminal electron accepting processes were determined. Sustained, anaerobic benzene biodegradation was obtained in transfer cultures containing less than 1% of the original soil inoculum. The rate of benzene degradation was variable, ranging from 1 μM/d to more than 75 μM/d. Growth of bacteria was linked to benzene degradation under sulphate-reducing and nitrate-reducing conditions. Growth was very slow, with doubling times of 9-30 days estimated by modelling benzene depletion curves to the Monod kinetic equation. The rate of benzene degradation was influenced most by biomass concentration and much less by the terminal electron accepting process.</p> <p> The ratio of moles of electron acceptor depleted to moles of benzene degraded was calculated and compared to the theoretically predicted ratios to confirm putative terminal electron acceptors. Anaerobic benzene degradation linked to iron reduction, sulphate reduction and methanogenesis was observed in enrichment cultures, corroborating results from previous studies. In addition, in some enrichment cultures, benzene degradation was linked to nitrate reduction. This is the first report demonstrating benzene degradation linked to nitrate reduction.</p> / Thesis / Master of Engineering (MEngr)

High performance liquid chromatography was used to measure ribonucleoside triphosphates in microbial samples. Anion exchange columns in a radial compression module were used to separate and quantify purine and pyrimidine ribonucleotides. Endogenous ribonucleoside triphosphates were extracted from Escherichia coli and pseudomonas aeruginosa using three different solvents, namely trifluorocetic acid (TFA; 0.5M), trichloroacetic acid (TCA; 6 per cent w/v) and formic acid (1.0M) Extracts were assayed for uridine 5'-triphosphate (ATP), and guanosine 5'-triphosphate (GTP) by using anion exchange radial compression high performance (pressure) liquid chromatography. The three extraction produres were compared for yield of triphosphates. E. coli, the TFA extraction procedure was more sensitive and reliable than TCA and formic acid extraction procedures, but , in P. aeruginosa, the best yields of ATP and GTP were obrained following extraction with TFA. Yields of UTP and CTP increased when extraction was performed in TCA. These data illustrate that different extraction produres produce different measures for different triphosphates, a point often overlooked.