Thesis (Ph.D. Biological Sciences)--University of Waikato, 2007. / Title from PDF cover (viewed March 6, 2008) Includes bibliographical references (p. 111-134)

Thesis (Ph.D.) - University of St Andrews, March 2008.

Thesis (M.S.)--University of North Carolina Wilmington, 2007. / Includes bibliographical references (leaves: 190-193)

The microbial ecology of 45 high temperature (> 50 ° C) petroleum reservoirs was investigated by isolating and characterizing bacteria that were present in their produced fluids. Initial work was aimed at selecting a suitable high temperature petroleum reservoir for the study of natural microbial populations. Experimental work then focussed on establishing the physico-chemical conditions that prevail in the selected reservoir and on developing media and enrichment conditions for the isolation of microorganisms indigenous to the reservoir. The ability of reservoir bacteria to grow and survive under the physical and chemical conditions found in the selected reservoir was used to assess the likelihood of an indigenous origin for these bacteria. The petroleum reservoir selected for study was the Alton petroleum reservoir (SW Queensland, Australia). It was established that most of the physico-chemical conditions in the Alton reservoir had remained unchanged since oil recovery began. The stability of redox conditions (90 mV) in the reservoir over its operating life was identified as an important factor in the coexistence of strict aerobic and strict anaerobic bacterial populations within the reservoir. An important change that has occurred in the Alton reservoir over its operating life because of oil recovery was an increase in water pH from 6.41 to 8.42 as a result of carbon dioxide loss (1.36 atm to 0.0134 atm) from the reservoir. Development of novel enrichment procedures that simulated Alton reservoir conditions led to the isolation of previously unreported aerobic and anaerobic populations of thermophilic bacteria. The aerobic bacteria isolated were identified as either endosporeforming heterotrophic bacteria from the genus Bacillus or nonspore-forming heterotrophic bacteria resembling members of the genus Thermoleophilum. All aerobes grew on carbon sources such as acetate and n-heptadecane that are normal constituents of the reservoir. The anaerobic bacteria isolated were characterized as sheathed fermentative bacteria from the order Thermotogales or non-sheathed fermentative bacteria. In parallel studies, the natural microbial populations in other reservoirs were investigated and I concluded that fermentative microorganisms were common inhabitants of high temperature petroleum reservoirs. The isolation of fermentative bacteria from these high temperature petroleum reservoirs established that fermentative bacteria are a fourth major microbial group, together with hydrocarbon-oxidizers, sulphate-reducers and methanogens, to be reported in petroleum reservoirs. The fermentative bacteria use organic nutrients and carbohydrates, but not contemporary crude oil as the principal nutrient source within reservoir waters. The thermophilic bacteria isolated from Alton petroleum reservoir demonstrated growth characteristics such as temperature (optima 50-70 ° C and range 37-85 ° C), pH (optima 6.0-9.0 and range 5.0-9.0 and salinity (optima 0-15 g per litre and range 0-30 g per litre), that were consistent with conditions encountered in the Alton reservoir (temperature 75 � C, pH 8.5 and TDS 2.7 g per litre). The isolated bacteria also demonstrated a number of characteristics that might enable them to survive adverse conditions that could be encountered in a petroleum reservoir environment. The characteristics that contribute to aerobic bacteria surviving in and overcoming periods of oxygen limitation include well-documented processes such as sporulation, by Bacillus spp., and microaerophily. The characteristics that contribute to fermentative bacteria surviving were: (1) a natural tolerance to reservoir physico-chemical fluctuations, (2) an ability to remain viable when metabolic activity was suppressed to very low rates by the growth-limiting conditions imposed, and (3) possible formation of viable ultramicrobacteria (UMB). Formation of UMB (bacteria smaller than 0.3 |im) by thermophilic bacteria has not been reported previously. The recovery of thermophilic UMB by filtration from the Alton reservoir water indicates that these bacteria occur in natural habitats. This study found the formation of thermophilic UMB and their survival characteristics differed considerably from that reported for the mesophilic, marine bacterium Vibrio sp. DWI. Unlike mesophilic marine bacteria, thermophilic bacteria did not always respond to nutrient deprivation by forming UMB and that these UMB did not show any increased ability to survive in the face of adverse conditions. Although the formation of UMB as part of routine cell growth and division was not demonstrated directly in this study, circumstantial evidence suggests that they form part of a natural life cycle. The exact conditions that result in UMB formation and their role in survival remain unresolved. The capacity of nonspore-forming indigenous populations from Alton to survive sudden shifts in environmental conditions that might result from common oilfield operations was poor. Such operations were demonstrated to be inhibitory or lethal to Alton reservoir bacteria. It also was concluded that such oilfield operations suppress indigenous microbiota. However, the impacts of most oilfield operations within a reservoir are likely to be confined to the immediate area surrounding injection and producing wells. Minimizing the localized effects of oilfield practices on indigenous reservoir populations will lead to the better management of undesirable microbial activity in reservoirs such as H2S formation (souring) and facilitate development of better microbially mediated oil recovery process. This study showed that selected reservoir isolates possess characteristics which are suitable for in situ biotechnological applications such as microbially enhanced oil recovery (MEOR). Characteristics favourable for enhanced oil recovery include a capability for UMB formation, which would enable better dispersion, and resistance to high concentrations of reservoir components such as calcium, magnesium, strontium, heavy metals and hydrocarbons.

Graduation date: 1994

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [87]-101).

Thesis (Ph.D.)--North Carolina State University. / Includes vita. Includes bibliographical references.

Thesis (Ph. D.)--Duke University, 2007. / Includes bibliographical references.

>Magister Scientiae - MSc / The negative environmental impact of fossil fuels and growing concerns about petroleum supplies has driven the search for alternative, renewable transportation fuels. An 'ideal' fuel replacement would be a biofuel produced from lignocellulosic biomass. Unfortunately, the presence of lignin in plant cell walls impedes the breakdown of cell wall polysaccharides into simple sugars and the subsequent conversion of these sugars into useable fuels. One of the most common fates of lignin in nature is to be metabolized by lignin peroxidases (LiPs), predominantly of microbial origin. This study aims to isolate and characterise microorganism(s) involved in the degradation of lignocellulose. Thermophilic bacteria were isolated from straw-based compost and screened for lignin peroxidase activity. One isolate, CP11, showed significant lignin peroxidase activity and based on 16S rRNA gene sequence analysis, the isolate was found to be most closely related to Bacillus thermoamylovorans. Morphological, physiological and biochemical characterisation was conducted to determine whether the isolate was a novel species. Morphologically, CP11 was characterised as an endospore-forming, Gram positive rod. In addition, the isolate was found to be a facultative anaerobe, catalase positive and capable of utilising a range of carbon sources including glucose, sucrose and arabinose. Isolate CP11 was moderately thermotolerant and grew between 37°C and 55°C, with an optimum growth temperature of 45°C. Based on its phenotypic characteristics CP11 could be clearly distinguished from its closest phylogenetic neighbours. Preliminary characterisation of the lignin peroxidase was conducted using crude enzyme extract and Azure B dye as the substrate. Activity was detected in the supernatant only and a growth curve was constructed to determine the growth phase of lignin peroxidase production. In order to identify the gene encoding the lignin peroxidase a small insert library was constructed and screened for ligninase activity using Azure B as the substrate. / National Research Foundation

This master thesis deals with conventional methods of bacterial cell analysis, polyhydroxyalkanoates, Raman spectroscopy and electron microscopy in the theoretical part. The production of polyhydroxybutyrate by selected thermophilic bacteria and their analysis by gas chromatography, cryogenic scanning electron microscopy and Raman spectroscopy is described in the experimental part. The chosen sample was analyzed by a transmission electron microscope. Comparing the results from previous mentioned methods it was found that the bacteria Schlegelella thermodepolymerans accumulated the highest amount of PHB. The lowest amount of PHB was obtained by bacteria Rubrobacter xylanophilus. The assumption that the PHB granules formed so-called needle-like plastic deformations during freeze-fracturing was affirmed by cryo-SEM photos analysis. Moreover, it was found that the bacterial cell characterization deduced from microscopic observation of samples corresponded to the description in the literature. TEM provided better resolution photos and in consequence the cells and PHB are more visible. The thesis is also focused on chemical fingerprint analysis of cells by Raman spectroscopy. Several biomolecules were identified by measured Raman spectra for the particular samples.