| dc.description.abstract | Canada is in the process of designing and implementing a deep geological repository (DGR) for long-term storage of used nuclear fuel. This proposed storage structure will be a multi-barrier system placed 500-800 metres belowground at a stable and informed host site within either a crystalline or sedimentary rock bed. Bentonite clay, which swells upon saturation, will be used to encase copper-coated used fuel containers and seal the DGR. The presence of naturally occurring microorganisms in the bentonite or subsurface environment, specifically anaerobic sulfate-reducing bacteria (SRB), is an important design consideration in the DGR due to their potential metabolic production of copper-corroding sulfides. For this reason, it is essential to study conditions for bentonite clay that will suppress microbial activity. Using pressure vessels for saturation, previous studies demonstrate that microbial growth is suppressed in compacted bentonite at swelling pressures over 2 MPa and water activities of 0.96 or less. After DGR establishment, the system will experience drying as radioactive heat dissipates, followed by a period of cooling and resaturation. Additionally, the salinity of groundwater penetrating the bentonite will vary depending on the chosen host rock. It is therefore important to investigate microbial responses to a variety of conditions in bentonite to account for possible combinations that will occur in the DGR and identify thresholds that suppress microbial proliferation. My thesis research explored the effects of water activity on Wyoming MX-80 bentonite clay in relation to microbial viability, abundance, and community composition, without the influence of pressure, by assessing microcosm samples in combinations of conditions using water activity, hydration solution, and oxygen presence. Microbial abundance estimates correlated with increasing water activity in oxic conditions. Highest overall microbial abundances were observed for oxic microcosms hydrated with SRB medium. Estimates for SRB remained unchanged for all conditions. No significant microbial proliferation occurred in microcosms hydrated with high-salinity medium or anoxic microcosms. These results suggest higher microbial abundances in the presence of high water activities and nutrient availability, and demonstrate suppression of microbial growth associated with both high-salinity hydration and anoxic incubation conditions. Microbial community profiles based on amplicon sequence variants resulting from direct 16S rRNA gene sequencing of oxic microcosms hydrated with water, low-salinity medium, or SRB medium revealed a dominance of Actinobacteriota, specifically ASVs affiliated with Saccharopolyspora in low and medium water activities and Streptomyces in medium and high water activities. These results indicate a growth dependence of specific actinobacterial taxa based on water activity. There were no consistent community composition pattern changes for anoxic microcosms or microcosms hydrated with high-salinity medium, consistent with a lack of growth based on cultivation-based and qPCR-based approaches. Quantification of fungal 18S rRNA genes from oxic samples hydrated with water or SRB medium revealed increasing abundances of fungi, specifically in microcosms hydrated with SRB medium. These data suggest that abundance estimates of aerobic heterotrophs from microcosms may include fungal colonies that match expected morphologies commonly observed on corresponding cultivation plates. Studying the effects of water activity and hydration media on bentonite is essential to understanding how microbial viability will be affected by water activity within a DGR environment, even in the absence of pressure. Overall, the results demonstrate that microbial growth is slowed by relatively low water activity, and that anoxic conditions and high-salinity water further suppresses bentonite-associated microbial growth. These results inform ongoing pressure vessel experiments that combine both water activity and pressure as controlling environmental parameters and will help guide DGR design specifications and the associated safety case for implementation. | en |
