Culturability and diversity of microorganisms recovered from an eight-year old highly-compacted, saturated MX-80 Wyoming bentonite plug

•Highly compacted bentonite (HCB) clay plug maintained under microbially-inhibiting conditions for ~ 8 years approached microbial background levels present in as-purchased bentonite clay.•Bacterial culturability results from long-term experiment agreed with previously conducted short-term experiments.•Microbial characterization of bacterial isolates revealed primarily Gram-positive spore-forming species.•Persistent (but reduced) bacterial culturability in HCB assumed to reflect in situ bacterial ability to form spores or transform to inactive state.

Highly-compacted bentonite (HCB)-based sealing systems are being developed for potential use in nuclear fuel waste repositories because of their inherent physicochemical and rheological properties. Results from previous short-duration (40–90 days) studies with highly-compacted (HC), water infused, confined Wyoming MX-80 bentonite revealed that a uniform dry density ≥  1.6 g/cm3 (resulting in a swelling pressure above 2000 KPa, water activity below 0.96 and an average pore size of < 0.02 μm) suppressed microbial culturability. A longer-term (7-year, 264-day) experiment using the same material and conditions was therefore conducted to determine whether these conclusions held over extended periods. Viable cell counts in material from the ~ 8-yr clay plug approximated those in the original uncompacted bentonite, confirming suppression of microbial growth. Bacteria recovered from the ~ 8 yr clay plug were comprised of primarily Gram-positive spore-formers. Together, these results confirmed that HCB suppressed microbial culturability (and presumably activity and growth) over extended time periods, and further revealed that the surviving culturable microorganisms were those adapted to life in low water activity soil environments.