Available at: http://digitalcommons.calpoly.edu/theses/456
Date of Award
MS in Biological Sciences
Christopher Kitts, Ph.D.
In an increasingly energy-hungry world, our capacity to meet the heightened energy demands of the future has become a pressing matter. The most urgent of these concerns are tied to the accessibility of petroleum. Various experts have proselytized both the imminent arrival of peak oil production rates and the ensuing decline of those rates thereafter. And to that end, the development of novel and advanced oil exploration methodologies has become almost as important as finding the sources of oil themselves.
The soils above petroleum reservoirs play host to various communities of alkane- oxidizing bacteria that can utilize the natural gas emitted by the reservoirs as a source of carbon and energy. While methane can originate from non-petroleum sources, the only natural sources of propane and butane are oil and gas fields. The increased presence of propane and butane-oxidizing bacteria in a given soil sample is used by oil prospectors as an accurate indicator of a proximal petroleum reservoirs.
For over a century, cell counts and hydrocarbon metabolic rates have been the metrics used to determine the presence of hydrocarbon-oxidizing microbes. These methods require weeks to complete. Here, we have developed a set of DNA primers for a much more rapid detection of hydrocarbon-oxidizing microbes through PCR amplification - for the chief purpose of petroleum exploration. Each primer’s design is based on a nucleotide sequence alignment of seven prmA and bmoX genes from seven organisms, which encode the large hydroxylase subunit of propane monooxygenase and the alpha hydroxylase subunit of butane monooxygenase respectively. These monooxygenases are the enzymes responsible for the initiation of propane and butane catabolism. Optimization of PCR with this primer set was accomplished using DNA extracted from known butane and propane oxidizers as positive controls, and methane and toluene oxidizers as negative controls. PCR products recovered from cultures of butane-oxidizing and propane-oxidizing bacteria, and soil samples, were sequenced. Phylogenetic trees were constructed from the sequencing data to confirm the accuracy of amplification. We demonstrate the use of PCR and agarose gel electrophoresis to detect hydrocarbon-oxidizing bacteria in culture and in complex microbial soil communities. Detection limits were elucidated through two different experiments. Potential avenues of advancements include narrowing specificity by selectively removing primer degeneracies, the use of additional positive and negative controls and the adaptation of the primers to a qPCR TaqMan assay.