The objective of this thesis was to characterize the microbiology of subsurface fracture water at a location within the Canadian Shield considered as a prospective host site for a geologic repository. Repository performance and long-term safety predictions for geologic confinement of radioactive waste rely on understanding the natural microbial processes that occur within a host formation; however, the inaccessibility of the crystalline terrestrial subsurface means that this habitat is difficult to explore and as such is largely unknown. The area characterized is located within the boundary of the Chalk River Laboratories site, situated within the Central Gneissic Belt of the Grenville province (formed 1.5 and 1.0 ba years before present); the site is also situated within the Ottawa-Bonnechere graben (formed 0.5 ba before present). Fracture water was accessed from one 34 m deep open drill hole and six cased and sealed drill holes; the Westbay Multilevel Groundwater Monitoring system preserved the natural fracture flow enabling discrete fracture water sampling at multiple depths via each of the sealed drill holes. This thesis combined multiple datasets in an exploratory analysis for drivers of subsurface microbial assembly within an ecological framework of selection, dispersal, drift and diversification across two spatial extents: 25 km3 and 1 km3. The outcomes of the multivariate analyses, null models and generalized linear models identified prospective source waters and distributional relationships of total and viable cell counts with fracture water sulfate and manganese. Random processes (dispersal, drift and diversification) explain close to 50% of the variance of the phylogenetic beta diversity among the component taxa. Selection associated with differential abundance of the 16S rRNA gene V4 region and spatial and environmental factors identified sulfate and organic carbon plus manganese and a spatial coefficient. Selection by differential abundance was not a major driver of community assembly; accounting for ~4% each of the total abundances linked to sulfate and manganese. At a spatial scale less than 1 km3, it may be possible to identify either greater significance for sulfate and manganese or to identify additional environmental factors linked to selection. Demonstrated metabolism included nitrate reduction (common across all sampling locations and at each sampling campaign) and sulfate reduction (observed at one sampling campaign). The distributions of total and viable cell counts correspond with the distributions of sulfate and manganese, respectively. The fracture water was a source of sulfate and manganese, but not a source of nitrate. A limited analysis of rock porewater identified sulfate and nitrogen compounds (ammonia, nitrite and nitrate) in parts per million concentrations, suggesting that the rock is a source of these compounds, and this finding warrants an assessment of rock weathering as a driver of selection at these sampling locations. Phylogenetic relationships across sampling locations showed that the component taxa were more closely related than expected by chance; this pattern suggests that, at the spatial scale of the analysis, competitive exclusion was not a driver of subsurface community assembly. Co-existence of close relatives may be biologically relevant—and thus be a sign of diversification—or this pattern may reflect the 16S rRNA gene copy number combined with intra-genomic heterogeneity greater than the 97% sequence similarity threshold for binning sequencing reads into organizational taxonomical units (OTUs). The distribution of genes for energy metabolism was uniform across all sampling locations. A metatranscriptome assessment would help differentiate between the genes present from the genes expressed. Testing for a wider range of demonstrated metabolic capabilities would support RNA-level gene expression analyses. Overall, applying the ecological framework of four main drivers of community assembly show that, at the spatial scale of the sampling, up to 50% of the variance among community dynamics reflect randomness. Approximately ~1% of the total abundance was linked to measured metabolism; at smaller spatial scales, ~8% of the total 16S rRNA gene abundance was linked to differential abundances across—potentially connected--sampling locations. By sampling at smaller spatial scales, therefore, it may be possible to discern additional metabolic and selective processes. These data will inform models for the performance and long-term safety of geological repositories.

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<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)