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.

This study was done to determine the effectiveness of a commercially available bioaugmentation product, BiOWiSHTM-Aqua FOG, for remediating petroleum-contaminated sandy soil. Biodegradation enhancement by BiOWiSHTM-Aqua FOG was evaluated in laboratory microcosms by directly measuring total petroleum hydrocarbon (TPH) and indirectly using respirometry. Attempts were made to enrich hydrocarbon-degrading bacteria in BiOWiSHTM-Aqua FOG, and the resulting enrichment cultures were screened using respirometry as well. Potential hydrocarbon-degrading bacteria in BiOWiSHTM-Aqua FOG were isolated. Experiments were performed at bench-scale using microcosm bottles containing sand contaminated with either motor oil or No. 2 diesel fuel. The microcosms were incubated at 25oC under aerobic conditions. TPH measurements of soil in the microcosms at 0, 25 and 56 days indicated that the addition of 500-ppm BiOWiSHTM-Aqua FOG improved biodegradation of the motor oil-contaminated soil by 45%. However, BiOWiSHTM-Aqua FOG did not have a measurable effect on biodegradation in the diesel-contaminated soil. In the respirometry experiments, BiOWiSHTM-Aqua FOG and two hydrocarbon-enriched BiOWiSHTM-Aqua FOG cultures were evaluated indirectly by the measurement of microbial carbon dioxide production and oxygen uptake using a MicroOxymaxTM respirometer. The respirometry experiments showed that in the six-day period following motor oil-contamination of soil, the addition of BiOWiSHTM-Aqua FOG substantially improves biodegradation rates. The added organisms in the product out-performed the indigenous organisms in the 5-6 days following contamination of the soil. The CO2 production observed in the BiOWiSHTM microcosms contaminated with motor oil was much greater than CO2 production without motor oil, which confirms that the observed metabolism can be attributed to motor oil biodegradation rather than metabolism of other organic material in the soil. Enriched consortia consistently generated far less CO2 than microcosms with the 500 ppm BiOWiSHTM-Aqua FOG. Stoichiometric calculations suggested that BiOWiSHTM-Aqua FOG removed approximately 1400 ppm TPH (14%) from the soil in 6.5 days, while an enrichment culture of BiOWiSHTM-Aqua FOG only reduced TPH levels by 459 ppm (5%). This result suggests that increased biodegradation rate in bioaugmented soil is aided by biodiversity in the augmenting inoculum. A potential hydrocarbon-degrading candidate organism was isolated from the product and cultured on Bushnell-Haas agar and plate-count agar (PCA). While at least two distinct colony types were successfully grown on media with motor oil, these same colonies appeared on Bushnell-Haas agar with no apparent carbon source, and survived repeated transfers onto this same medium. Therefore, their status as hydrocarbon-degraders is inconclusive. More thorough enrichment work could be pursued, especially using soil samples collected from petroleum-contaminated sites.

Soil organic carbon (SOC) is concentrated in coastal wetlands, and its permanence maintains a livable climate, yet dynamics that govern microbial activity and SOC persistence are not fully characterized in coastal wetlands. Though microbial activity is conventionally thought to facilitate SOC loss, soil microbes simultaneously direct SOC formation. In fact, microbially-processed materials constitute up to half of the terrestrial soil organic carbon pool. Environmental conditions can affect whether microbes yield a net gain or loss of SOC, yet there is little consensus on microbial drivers of soil carbon longevity in coastal ecosystems. I sought to identify which drivers of microbial activity have the greatest impact on SOC in salt marsh soils. To address this question, I conducted a meta-analysis using the PRISMA method. Based on an initial survey of 2,835 studies, numeric data on soil and ecosystem characteristics were collected across 50 studies on over 60 salt marshes located around the world. Integrative data analyses, including structural equation modeling (SEM), were applied to synthesized data to identify environmental drivers of SOC in salt marsh ecosystems. Across a wide range of study sites, analysis of over 20 variables shows that soil characteristics are tightly linked. Salinity, pH, nitrogen, and phosphorus are associated with increased microbial biomass and soil organic carbon. Correlations between microbial biomass carbon and SOC are strengthened by soil salinity and nitrogen, and they are weakened by moisture. Correlations were dependent on the means by which variables were measured, yet findings were consistent across study sites. These results suggest that soil carbon content is affected by drivers of microbial activity. Observational findings set the stage for experimental strategies that parcel causal effects of microbial activity on SOC from confounding effects of covariant environmental conditions. I identified that nitrogen, phosphorus, salinity, pH, and moisture influence microbial contributions to SOC. These environmental drivers, as well as microbial biomass and greenhouse gas flux, should be considered key indicators of soil health when assessing the sustainability of coastal SOC. Identification of environmental drivers of microbial function enables design of land management strategies that promote conditions conducive to coastal soil carbon longevity.

Foraminifera are a diverse clade of mostly shell-building single-celled organisms. Estimation of foraminiferal diversity is critical for understanding past and present climatic conditions, as they are highly sensitive to environmental perturbations. Biodiversity estimates of foraminifera began with the counting of test (i.e., shell) microfossils composed of calcium carbonate, as they are well preserved in sediment samples. However, this view has changed with molecular biodiversity estimates, which suggest that early-diverging single-chamber (i.e., "monothalamid") species that lack preservation ability are more diverse than anticipated. Although biodiversity estimates of foraminifera at the molecular level have changed our perceptions, they possess various challenges, especially with metabarcoding approaches. The metabarcoding approach is challenging in foraminifera because small subunit ribosomal (SSU) rRNA gene does not PCR amplify "universal" eukaryotic primers due to the presence of large insertions. Therefore, studies of foraminiferal diversity require targeted primers. Similarly, the pair-wise sequence similarity approach to taxonomic resolution can be problematic for Foraminifera, as fewer matching reference database exists for “monothalamids”- this requires the use of a more robust phylogeny-informed taxonomy, which provides a taxonomic identification for each sequence. Also, the appropriateness of recently developed metabarcoding tools still needs validation and comparison with clustering approaches for foraminiferal biodiversity estimation. This chapter introduces the current state of knowledge of foraminiferal biodiversity while also describing the knowledge gaps addressed in this thesis.

Viruses hold our attention for the horrific impact they have on human health and welfare. However, viruses are a critical part of our ecosystem and facilitate the cycling of carbon and other important nutrients. The cycle of virus infection, followed by host resistance and the subsequent evolution of new strains enables adaptation to changing hosts and the environment. Giant viruses, some with particle sizes large enough to be visible in light microscopes and their bewildering array of accessory genes, have captivated scientists and the general public since their discovery two decades ago. Giant viruses are part of the Nucleocytoviricota (NCV) whose members include both harmful agents (such as the causative agents of smallpox and swine hemorrhagic fever) and beneficial ones (such as those that provide biocontrol of insects, mitigation of toxic algal blooms and enzymes for biotechnology). Most of the giant viruses discovered, to date, are from marine and freshwater ecosystems where their hosts are abundant. In terrestrial soils, very few giant viruses have been revealed because of challenges in shifting through the astounding microbial genetic diversity in soil to assemble genomes from metagenomic data. Currently there is a lack of knowledge about abundance and genetic diversity of giant viruses in terrestrial soils, knowledge about their hosts and their influence on biogeochemical cycling. In 2018, giant viruses were discovered in the Barre Woods experimental warming plots at Harvard Forest. (Schulz et al 2018 Nature Communications). A novel environmental genomics approach involving filtration and fluorescence activating cell-sorting (FACS) was used to discover 16 Nucleocytoviricota (NCVs) in just a few grams of Harvard Forest soil. All these newly discovered viruses represent distinct lineages (new species, genera, and families). This experiment involved just two soil cores (1 warming and 1 control) and a single time point at Harvard Forest. There is much to learn about the terrestrial giant virus genetic biodiversity as these same viruses have not yet been discovered at other sites around the world. My research will focus on a genus of giant viruses with only three known representatives, all from Harvard Forest. They are Hyperionvirus (with the world’s 2nd largest virus genome at 2.4 MBp), Terrestrivrus the 10 th largest genome at 1.8 MBp), and Harvfovirus (the 15 th largest genome at 1.6 MBp). In the experimental warming plots the relative amount of bacteria to fungi has increased. We hypothesize that the relative increase in bacteria has led to an increase in protists, which feed on the bacteria, which in turn has led to an increase in giant viruses, which infect the protists. Because of the high genetic diversity in viruses and the lack of ribosomal genes, it is not possible to create primers that span the entire Nucleocytoviricota phylum or even at the family level. To test our hypothesis, we designed degenerate PCR primers that detect and quantify members of the genus containing the 3 giant Harvard Forest viruses. DNA was extracted from soil samples the soil (stored at -80C) from the 2017 temperature toggle experiment at Barre Woods in which the power to the warmed plots was turned off from late May until early September were used. The giant viruses were originally discovered in the sample just prior to turning off the power. We used 4 time points spanning the experiment with 8 samples from each the warmed and control plots (4 x 16 = 16 samples total). The primers were designed based on five hallmark genes that are present in most members of the Nucleocytoviricota. After amplification, the amount of DNA would be quantified and normalized. We expect to better understand the genetic diversity of this genus of giant viruses in the soil including the possibility of detecting new species in this genus.

Vibrio choleraeis the etiological agent of the severe diarrheal disease-cholera and natural inhabitant of estuarine and coastal waters. The proximity of the Florida Indian River Lagoon (IRL) to areas affected by recent cholera outbreaks makes this estuary ideal to investigate the environmental dynamics and their potential role in V. cholerae's pathogen emergence. We identified two locations in the IRL, Feller's House UCF Field Station and Shepard Park, as our collection sites. We collected samples from three different fractions - water, plankton, and sediment - and recorded data for several water parameters such as pH, temperature, and, turbidity. In the laboratory, we enriched samples in alkaline peptone water and isolated V. choleraeusing widely used selective media Thiosulfate-Citrate Bile Salts-sucrose agar (TCBS) and CHROMagar Vibrio. From our study, we isolated 100 potential V. cholerae isolates, which were confirmed using biochemical tests such as oxidase and Kligler's Iron Agar. V. cholerae has allelic variations in the core genes such as ompU, which provide pre-adaptation to virulence. We investigated the allelic variations within ompU to characterize V. cholerae isolates. We elucidated the sequenced allele of ompU and built a neighboring-joining phylogeny tree to view the differentiation among vibrios. Our findings provide insight into the endemic population of V. cholerae in the Eastern Coast of Florida. Further studies include a screen for additional virulence genes and investigate the role of environmental dynamics on the distribution of V. cholerae and emergence as a human pathogen.

The ecology and taxonomy of the snowbank fleshy fungi in the vicinity of Mirror Lake, Uinta Mountains, Utah were investigated during 1963, 1964, and 1965. A total of 59 speciaes, representing the Agaricales, Polyporales, Thelephorales, Gastromycestes, Myxomycetes, Dacrymycetales, and Ascomycetes, were found associated with the snowbanks. Four of these species (Geupiniopsis alpinus, Polyporus alboluteus, Polyporus leucospongia and Sterile myclium I) have presence percentages of 100% and three (Stereum Rugisporum, Dasyscypha arida and Herpotichia nigra) had presence percentages of 90%. The lignicolous fungi were found to be more frequent around the snowbanks than either the terrestrial or coprophilous fungi. No fleshy fungi were found fruiting in association with the snowbanks in wet meadows. All terrestrial fungi found in association with the snowbanks were found either around snowbanks in the forest or in the transitional zone between the forest and the wet meadows. Twenty-two species began their development under the snow. Sporocarps kept buried in the snow did not develop a mature hymenium while in the snow, whereas similar sporocarps where the snow was allowed to melt developed mature hymenia in the same period of time.

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<p>Recreational beaches are important local resources for attracting tourists. It is critical to keep tracking recreational water quality to prevent public health issues. Waterborne pathogens are one of the main elements that could cause recreational water related diseases. Fecal pollution is the primary source of waterborne pathogens. Therefore, it is important to quantify the amount of fecal pollution indicators that are present in the water, particular the human fecal indicator. The primary objective of this thesis is to develop an integrative microbial quality monitoring system to better understand water quality. The first part of this thesis examined the presence of a general fecal pollution indicator (<em>E. coli</em>) and a human fecal pollution indicator (human-specific <em>Bacteroidales</em>). The correlations between pollution sources and beach water quality were also studied to identify the impact of pollution sources. The results revealed the highly localized correlations at individual beaches depended on the impact from pollution sources. The weak correlations suggested some previous assumed pollution sources may only weakly impacted beach water quality.</p> <p>Because <em>E. coli</em> strains differ enormously in pathogenic potential, it is possible that environmental <em>E. coli</em> have different genetic compositions and differential gene expression in genes such as the global stress regulator <em>rpoD</em> and <em>rpoS</em>. Thus, the second part of this thesis examined genetic composition and gene expression in <em>E. coli</em> environmental strains to study how global gene expression is altered in the natural environment. The results revealed differential RpoSexpression levels in environmental <em>E. coli </em>strains, suggesting that genes regulated by <em>rpoD</em> and <em>rpoS</em> may have differential expression levels in environmental strains, compared to commonly studied laboratory strains.</p> / Master of Science (MSc)