Stability of Microbial Community Profiles Associated with Compacted Bentonite from the Grimsel Underground Research Laboratory

<jats:title>ABSTRACT</jats:title>
<jats:p>To assess the microbiology and corrosion potential of engineered components of a deep geological repository for long-term storage of high-level nuclear waste, the Materials Corrosion Test is being conducted at the Underground Research Laboratory in Grimsel, Switzerland. Modules containing metal coupons surrounded by highly compacted MX-80 bentonite, at two dry densities (1.25 and 1.50 g/cm<jats:sup>3</jats:sup>), were emplaced within 9-m-deep boreholes, and the first modules were retrieved after 13 months of exposure. Bentonite and associated module materials were sampled, and microbial communities and their distributions were assessed using 16S rRNA gene sequencing and phospholipid fatty acid (PLFA) analysis. Borehole fluid was dominated by amplicon sequence variants (ASVs) affiliated with <jats:italic>Desulfosporosinus</jats:italic> and <jats:italic>Desulfovibrio</jats:italic>, which are putatively involved in sulfate reduction. The relative abundance of these ASVs was lower for samples from inside the borehole module, and they were almost undetectable in samples of the inner bentonite layer. The dominant ASV in case and filter sample sequence data was affiliated with <jats:named-content content-type="genus-species">Pseudomonas stutzeri</jats:named-content>, yet its relative abundance decreased in the inner layer samples. <jats:italic>Streptomyces</jats:italic> sp. ASVs were relatively abundant in all bentonite core sample data both prior to emplacement and after 13 months of exposure, presumably as metabolically inactive spores or extracellular “relic” DNA. PLFA concentrations in outer and inner layer bentonite samples suggested cellular abundances of 1 × 10<jats:sup>6</jats:sup> to 3 × 10<jats:sup>6</jats:sup> cells/g, with similar PLFA distributions within all bentonite samples. Our results demonstrate consistent microbial communities inside the saturated borehole module, providing the first evidence for microbial stability under conditions that mimic a deep geological repository.</jats:p>
<jats:p><jats:bold>IMPORTANCE</jats:bold> The Materials Corrosion Test in Grimsel Underground Research Laboratory, Switzerland, enables an evaluation of microbiological implications of bentonite clay at densities relevant for a deep geological repository. Our research demonstrates that after 13 months of exposure within a granitic host rock, the microbial 16S rRNA gene signatures of saturated bentonite clay within the modules were consistent with the profiles in the original clay used to pack the modules. Such results provide evidence that densities chosen for this emplacement test are refractory to microbial activity, at least on the relatively short time frame leading to the first time point sampling event, which will help inform <jats:italic>in situ</jats:italic> engineered barrier system science. This study has important implications for the design of deep geological repository sites under consideration for the Canadian Shield.</jats:p>