One of the major challenges for future Deep Geological Repositories (DGRs) for radioactive waste is to ensure long-term safety over timescales exceeding 100,000 years. The effect of gamma radiation on bentonite microorganisms remains poorly understood, with even less research exploring its combined impact with other key repository conditions. This study investigates the combined effects of high bentonite compaction density (1.6 g/cm3) and gamma radiation (cumulative doses 14 kGy or 28 kGy) on FEBEX bentonite blocks under complete water saturation with synthetic pore water, anoxic atmosphere, and sulfate-reducing bacteria (SRB). Culture-dependent microbiological analyses and next-generation sequencing demonstrated that native FEBEX bentonite microorganisms can withstand these challenging conditions, favoring spore-forming bacteria. After 1-year incubation, in both non-irradiated and irradiated samples, extremophile genera like Saccharopolyspora and Streptomyces, were identified. In addition, gamma radiation negatively impacted heterotrophic aerobe viability. However, a key finding was that a 6-month incubation period prior to radiation exposure enhanced microbial resistance to this stressor. Mineralogically, neither gamma radiation nor other studied conditions affected the properties of FEBEX bentonite, which remained stable. This study provides groundbreaking insights into the combined effects of gamma radiation and other DGR-relevant conditions on bentonite microbiology and mineralogy, significantly advancing the field.

Division of Materials of Energy Interest CIEMAT Avenida Complutense 40 Madrid 28040 Spain

Faculty of Sciences Department of Microbiology University of Granada Granada Spain

Faculty of Sciences Department of Microbiology University of Granada Granada Spain; Current address Helmholtz Zentrum Dresden Rossendorf e 5 Institute of Resource Ecology Bautzner Landstrasse 400 Dresden 01328 Germany

Nuclear Fission Department CIEMAT Avenida Complutense 40 Madrid 28040 Spain

Technical University of Liberec Institute for Nanomaterials Advanced Technologies and Innovations Bendlova 1407 7 Liberec 461 17 Czech Republic

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