<p>Investigating the presence of microbial communities in the Earth's deep terrestrial subsurface and the metabolic processes taking place in these environments provides insight into the some of the ultimate limits for life on Earth, as well as the potential for microbial life to exist within the subsurface of other planetary bodies. This Master's thesis project utilized phospholipid fatty acid (PLFA) analysis, in combination with carbon isotope analyses (δ¹³C and Δ¹⁴C), to explore the presence and activity of microbial communities living within deep terrestrial subsurface fracture water systems and low permeability, deep sedimentary rocks. Deep fracture water systems, ranging from 0.9 to 3.2 km below land surface, were sampled for microbial communities via deep mine boreholes in the Witwatersrand Basin of South Africa. PLFA concentrations revealed low biomass microbial communities, ranging from 2x10¹ to 5x10⁴ cells per mL and the PLFA profiles contained indicators for environmental stressors, including high temperatures and nutrient deprivation. δ¹³C and Δ¹⁴C analyses of PLFAs and potential carbon sources (dissolved inorganic carbon (DIC), dissolved organic carbon (DOC) and methane) identified microbial utilization of methane in some systems and utilization of DIC in others. Evidence for microbial oxidation of methane and chemoautotrophy in these systems is consistent with a self-sustaining deep terrestrial subsurface biosphere that is capable of surviving independent of the photosphere. Viable microbial communities were also identified within deep (334 to 694 m depth) sedimentary rock cores sampled from the Michigan Basin, Canada. PLFA analyses revealed microbial cell densities ranging from 1-3 x 10⁵ cells/mL and identified PLFA indicators for environmental stressors. These results demonstrate the ubiquity of microbial life in the deep terrestrial subsurface and provide insight into microbial carbon sources and cycling in deep microbial systems which may persist in isolation over geologic timescales.</p> / Master of Science (MSc)

Investigation of life’s limitations on Earth provides the necessary information to constrain where life outside of Earth may be proliferating or previously existed. This Master’s thesis applied phospholipid fatty acid (PLFA) analysis in combination with organic carbon and 16S rRNA gene data to assess and characterize microbial communities through both microcosms and in situ samples of bentonite clay, which is an intended barrier component for the long-term storage of high-grade nuclear waste. Microcosm experiments were set up to test the impact of water activity in as-received, uncompacted bentonite clays using a high (0.99) and low (0.93) water activity over a one month period. Under aerobic incubation water activities of 0.93 and 0.99 had no resolvable effect between water activity levels on the growth of cells of indigenous communities of microbes in as-received uncompacted bentonite. Growth was detected under both water activities by a significant increase in total PLFA abundance. The increase in PLFA over the period of the study suggested an approximate increase in cells from 4x10^6 to 2x10^7 E.coli equivalent cells/g. The distribution of the PLFA and genetics data suggests the community is composed predominantly of gram-positive aerobic heterotrophs with lesser amounts of anaerobic bacteria and eukaryotes potentially in the form of fungi. Similar cell abundances and community structures were identified in the Tsukinuno Mine bentonite DGR analogue site which is a ~12 to 16 Ma deposit approximately 200 m below the surface. Total PLFA recovered from the core subsamples ranged from 32 pmol PLFA/g to 431 pmol PLFA/g, which corresponds to a range from 7.5x10^5 to 1.2x10^7 E.coli equivalent cells/g, across all cores. The community was composed of both aerobic and anaerobic bacteria consisting of gram-positive and gram-negative bacteria, as well as possible sulfate-reducing bacteria and eukaryotes. / Thesis / Master of Science (MSc)