Effect of temperature on microbial communities in bentonite for use in engineered barrier systems

Bentonite is an important component of deep geological repository (DGR) designs, where it will serve as a buffer between used fuel containers (UFCs) and subsurface rock walls of the repository. The potential for microbial activity in bentonite is being studied to understand the influence of microbial metabolisms (e.g., sulfate reduction by sulfate-reducing bacteria) on the long-term safety of the DGRs (e.g., through contributions to microbiologically influenced corrosion). Most studies of microorganisms in bentonite involved culturing microorganisms at 30°C or below, even though the placement room in a DGR is expected to experience elevated temperatures for up to one thousand years after used fuel placement. The purpose of this study was to test the abundance and community composition of microorganisms in as-received and hydrated bentonite at a range of DGR-relevant temperatures (15-105°C) using a combination of cultivation and DNA-based techniques. In certain clays tested, aerobic heterotrophs, anaerobic heterotrophs, and sulfate-reducing bacteria were culturable at 15, 30, 45, and 60°C from both the as-received and hydrated bentonite, demonstrating that the bentonite microbial community includes representatives capable of growth at a range of DGR-relevant temperatures. Although cultivation results showed no significant increase in the abundance of culturable microorganisms from as-received bentonite to hydrated bentonite at temperatures greater than 45°C, sequencing results for two bentonite samples hydrated and incubated at 60°C revealed that 16S rRNA gene profiles were dominated (>99%) by sequences associated with the putative thermophilic family Thermoactinomycetaceae, which was not detectable in the as-received bentonite starting material. Not only does this suggest that as-received bentonite harbours viable thermophiles, but it also highlights the importance of a multifaceted (e.g., cultivation coupled to DNA sequencing) approach to study microbial communities of bentonite.IMPORTANCEPredicting the abundances and types of microorganisms that may be active within a deep geological repository is critical to ensure that DGR design specifications minimize or prevent microbially mediated deterioration of DGR components. To date, research in this area has focused on the effect of bentonite dry density and the associated swelling pressure on suppression of microbial growth, but most of these experiments have been conducted at relatively low temperatures (e.g., 30°C). Studying the microbiology of bentonite exposed to elevated temperatures is critical given that a DGR is expected to experience high temperatures for up to one thousand years.