Microbial growth inhibition by compacted bentonite after an 8.5-year in-situ incubation

Compacted bentonite in future deep geological repositories for the disposal of nuclear waste will create an extreme, energy-limited environment for microorganisms, yet the long-term implications for microbial community structure and the potential emergence of sulfate-reducing bacteria (SRB) remain uncertain. Here, we use an 8.5-year in-situ incubation experiment in an anoxic Opalinus Clay borehole, integrating observations from successive retrieval phases after 1.5, 5.5, and 8.5 years, to examine changes in microbial persistence in compacted Wyoming bentonite across three dry densities (1.25, 1.45, and 1.55 g cm −3 ). 16S rRNA gene quantification and sequencing showed that microbial cell numbers remained low and declined significantly over time at all densities, with density as the primary driver and time as the dominant secondary driver of community change. After 8.5 years, SRB were detected only in the rare biosphere (<0.1% relative abundance), although they were recoverable by cultivation, indicating survival rather than growth. Altogether, these results support limited concern about the progressive enrichment of corrosion-inducing microorganisms following oxygen depletion and demonstrate that bentonite compaction at 1.25 g cm −3 is sufficient to inhibit microbial growth, providing an effective biological barrier.