Low water activity limits bentonite-associated microbial growth

AIMS: This research investigated the impact of water activity on microbial abundance estimates from bentonite under conditions relevant to a deep geological repository for used nuclear fuel. Because previous research tested saturation of bentonite within pressurized vessels, the goal of this study was to assess how water activity alone, without pressure, prevents increases in microbial abundance estimates from bentonite samples. METHODS AND RESULTS: Small-scale microcosms of bentonite were hydrated to low, medium, or high water activities, with Type I water, reference groundwater, or bacterial growth medium, then incubated under oxic or anoxic conditions. At six timepoints over a 6-month period, microorganisms were quantified using cultivation-dependent and independent approaches, and 16S rRNA genes were sequenced to monitor relative abundance changes of microbial taxa. Large-scale incubations were then conducted to also enable analyses of phospholipid fatty acids and natural organic matter. The results demonstrate that increasing water activity was associated with higher microbial abundance estimates for oxic condition incubations, with water-activity-dependent actinobacterial growth. In contrast, no significant microbial abundance changes were observed for anoxic microcosm incubations. For all tested conditions, we did not detect any increases in sulfate-reducing bacteria abundance estimates. CONCLUSIONS: Although low water activity conditions prevented changes in microbial abundances for bentonite samples incubated under oxic conditions, anoxic conditions alone prevented increases in abundances of culturable microorganisms. These results complement previous pressure-vessel studies that have shown how low water activity and elevated pressure simultaneously reduce the abundance of viable microorganisms that can initially proliferate during the saturation process.