As bentonite hosts a diverse spectrum of indigenous microorganisms with the potential to influence the long-term stability of deep geological repositories, it is essential to understand the factors influencing microbial activity under repository conditions. Here, we focus on two factors, i.e., temperature and swelling pressure, using a suspension of Cerny Vrch bentonite to boost microbial activity and evaluate microbial response. Suspensions were exposed either to different pressures (10, 12 and 15 MPa; to simulate the effect of swelling pressure) or elevated temperatures (60, 70, 80 and 90 °C; to simulate the effect of cannister heating) for four weeks. Each treatment was followed by a period of anaerobic incubation at atmospheric pressure/laboratory temperature to assess microbial recovery after treatment. Microbial load and community structure were then estimated using molecular-genetic methods, with presence of living cells confirmed through microscopic analysis. Our study demonstrated that discrete application of pressure did not influence on overall microbial activity or proliferation, implying that pressure evolution during bentonite swelling is not the critical factor responsible for microbial suppression in saturated bentonites. However, pressure treatment caused significant shifts in microbial community structure. We also demonstrated that microbial activity decreased with increasing temperature, and that heat treatment strongly influenced bentonite microbial community structure, with several thermophilic taxa identified. A temperature of 90 °C proved to be limiting for microbial activity and proliferation in all bentonite suspensions. Our study emphasizes the crucial role of a deep understanding of microbial activity under repository-relevant conditions in identifying possible strategies to mitigate the microbial potential within the deep geological repository and increase its long-term stability and safety.

Disposal processes and safety ÚJV Řež a s Hlavní 130 250 68 Husinec Czech Republic

Institute for Nanomaterials Advanced Technologies and Innovation Technical University of Liberec Bendlova 7 460 01 Liberec Czech Republic

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Aoki K, Sugita Y, Chijimatsu M, Tazaki K. Impacts of thermo-hydro-mechanical experiments on the microbial activity in compacted bentonite at the Kamaishi mine, Northeast Japan. Appl Clay Sci. 2010;47:147–154. doi: 10.1016/j.clay.2008.12.016. DOI

Baik M-H, Cho W-J, Hahn P-S. Erosion of bentonite particles at the interface of a compacted bentonite and a fractured granite. Eng Geol. 2007;91:229–239. doi: 10.1016/j.enggeo.2007.02.002. DOI

Bengtsson A, Pedersen K. Microbial sulphate-reducing activity over load pressure and density in water saturated Boom Clay. Appl Clay Sci. 2016;132–133:542–551. doi: 10.1016/j.clay.2016.08.002. DOI

Bengtsson A, Pedersen K. Microbial sulphide-producing activity in water saturated Wyoming MX-80, Asha and Calcigel bentonites at wet densities from 1500 to 2000 kg m–3. Appl Clay Sci. 2017;137:203–212. doi: 10.1016/j.clay.2016.12.024. DOI

Bennett D, Gens R. Overview of European concepts for high-level waste and spent fuel disposal with special reference waste container corrosion. J Nucl Mater. 2008;379:1–8. doi: 10.1016/j.jnucmat.2008.06.001. DOI

Bokulich NA, Kaehler BD, Rideout JR, et al. Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2’s q2-feature-classifier plugin. Microbiome. 2018;6:90. doi: 10.1186/s40168-018-0470-z. PubMed DOI PMC

Bolyen E, Rideout JR, Dillon MR, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol. 2019;37:852–857. doi: 10.1038/s41587-019-0209-9. PubMed DOI PMC

Brown AR, Boothman C, Pimblott SM, Lloyd JR. The impact of gamma radiation on sediment microbial processes. Appl Environ Microbiol. 2015;81:4014–4025. doi: 10.1128/AEM.00590-15. PubMed DOI PMC

Callahan BJ, McMurdie PJ, Rosen MJ, et al. DADA2: high resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13:581–583. doi: 10.1038/nmeth.3869. PubMed DOI PMC

Claesson MJ, Wang Q, O’sullivan O, et al. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res. 2010;38:e200–e200. doi: 10.1093/nar/gkq873. PubMed DOI PMC

Clifford RJ, Milillo M, Prestwood J, et al. Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR. PLoS ONE. 2012;7:e48558. doi: 10.1371/journal.pone.0048558. PubMed DOI PMC

Dohnálková M, Vondrovic L, Hausmannová L (2022) Technické řešení hlubinného úložiště. SÚRAO

Dowd SE, Callaway TR, Wolcott RD, et al. Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) BMC Microbiol. 2008 doi: 10.1186/1471-2180-8-125. PubMed DOI PMC

Dunn PH, Barro SC, Poth M. Soil moisture affects survival of microorganisms in heated chaparral soil. Soil Biol Biochem. 1985;17:143–148. doi: 10.1016/0038-0717(85)90105-1. DOI

Engel K, Coyotzi S, Vachon MA, et al. Validating DNA extraction protocols for bentonite clay. mSphere. 2019;4(5):10–1128. doi: 10.1128/mSphere.00334-19. PubMed DOI PMC

Feiveson M, Ramana H (2011) Managing spent fuel from nuclear power reactors. Experience and lessons from around the world

Gilmour KA, Davie CT, Gray N. Survival and activity of an indigenous iron-reducing microbial community from MX80 bentonite in high temperature / low water environments with relevance to a proposed method of nuclear waste disposal. Sci Total Environ. 2022 doi: 10.1016/j.scitotenv.2021.152660. PubMed DOI

Grigoryan AA, Jalique DR, Medihala P, et al. Bacterial diversity and production of sulfide in microcosms containing uncompacted bentonites. Heliyon. 2018 doi: 10.1016/j.heliyon.2018.e00722. PubMed DOI PMC

Hlavackova V, Shrestha R, Hofmanova E, et al. A protocol for the extraction of viable bacteria for identification of bacterial communities in bentonite. Appl Clay Sci. 2023 doi: 10.1016/j.clay.2022.106809. DOI

Hökmark H. Hydration of the bentonite buffer in a KBS-3 repository. Appl Clay Sci. 2004;26:219–233. doi: 10.1016/j.clay.2003.12.034. DOI

Johnson LH, Niemeyer M, Klubertanz G et al (2002) Calculations of the temperature evolution of a repository for spent fuel, vitrified high-level waste and intermediate level waste in Opalinus Clay. National Cooperative for the Disposal of Radioactive Waste (NAGRA)

Kašpar V, Šachlová Å, Hofmanová E et al (2021) Geochemical, geotechnical, and microbiological changes in Mg/Ca bentonite after thermal loading at 150°C. Minerals 11:965. 10.3390/min11090965

Kim J, Dong H, Seabaugh J, et al. Role of microbes in the smectite-to-illite reaction. Science. 2004;303:830–832. doi: 10.1126/science.1093245. PubMed DOI

King F, Kolář M. Lifetime predictions for nuclear waste disposal containers. Corrosion. 2018;75:309–323. doi: 10.5006/2994. DOI

Lhotský O, Kukačka J, Slunský J, et al. The effects of hydraulic/pneumatic fracturing-enhanced remediation (FRAC-IN) at a site contaminated by chlorinated ethenes: a case study. J Hazard Mater. 2021;417:125883. doi: 10.1016/j.jhazmat.2021.125883. PubMed DOI

Lydmark S, Pedersen K. Äspö Hard Rock Laboratory. Canister Retrieval Test. Microorganisms in buffer from the Canister Retrieval test – numbers and metabolic diversity. Stockholm, Sweden: Swedish Nuclear Fuel and Waste Management Co; 2011.

Madigan MT, Bender KS, Buckley DH et al (2018) Brock biology of microorganisms, Global Edition. New York

Martinez Abizu P (2020) PairwiseAdonis: pairwise multilevel comparison using adonis. R Package Version 0.4

Masurat P, Eriksson S, Pedersen K. Microbial sulphide production in compacted Wyoming bentonite MX-80 under in situ conditions relevant to a repository for high-level radioactive waste. Appl Clay Sci. 2010;47:58–64. doi: 10.1016/j.clay.2009.01.004. DOI

Matschiavelli N, Kluge S, Podlech C, et al. The year-long development of microorganisms in uncompacted bavarian bentonite slurries at 30 and 60°C. Environ Sci Technol. 2019;53:10514–10524. doi: 10.1021/acs.est.9b02670. PubMed DOI

McMurdie PJ, Holmes S. Phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE. 2013;8:e61217. doi: 10.1371/journal.pone.0061217. PubMed DOI PMC

Merroun ML, Raff J, Rossberg A, et al. Complexation of uranium by cells and S-Layer sheets of Bacillus sphaericus JG-A12. Appl Environ Microbiol. 2005;71:5532–5543. doi: 10.1128/AEM.71.9.5532-5543.2005. PubMed DOI PMC

Miettinen H, Bomberg M, Bes R, et al. Transformation of inherent microorganisms in Wyoming-type bentonite and their effects on structural iron. Appl Clay Sci. 2022;221:106465. doi: 10.1016/j.clay.2022.106465. DOI

Miettinen H, Bomberg M, Vikman M (2015) Microbial activation due to addition of electron donors/acceptors in deep groundwaters. MIND

Němeček J, Dolinová I, Macháčková J, et al. Stratification of chlorinated ethenes natural attenuation in an alluvial aquifer assessed by hydrochemical and biomolecular tools. Chemosphere. 2017;184:1157–1167. doi: 10.1016/j.chemosphere.2017.06.100. PubMed DOI

Nicholson WL, Munakata N, Horneck G, et al. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev. 2000;64:548–572. doi: 10.1128/MMBR.64.3.548-572.2000. PubMed DOI PMC

Otte JM, Blackwell N, Soos V, et al. Sterilization impacts on marine sediment-are we able to inactivate microorganisms in environmental samples? FEMS Microbiol Ecol. 2018;94:fiy189. doi: 10.1093/femsec/fiy189. PubMed DOI

Posiva Oy (2013) Safety case for the disposal of spent nuclear fuel at Olkiluoto—performance assesment 2012. POSIVA 2012-04. POSIVA

Posiva Oy (2017a) Safety evaluation for a KBS-3H spent nuclear fuel repository at Olkiluoto—features, events and processes. POSIVA 2016-03. POSIVA

Pedersen K (2017) Bacterial activity in compacted bentonites. MIND

Pedersen K, Motamedi M, Karnland O, Sandén T. Mixing and sulphate-reducing activity of bacteria in swelling, compacted bentonite clay under high-level radioactive waste repository conditions. J Appl Microbiol. 2000;89:1038–1047. doi: 10.1046/j.1365-2672.2000.01212.x. PubMed DOI

Posiva Oy SKB (2017) Safety functions, performance targets and technical design requirements for a KBS-3V repository—conclusions and recommendations from a joint SKB and Posiva working group. Posiva SKB Report 01. POSIVA, SKB

Potts M. Desiccation tolerance of prokaryotes. Microbiol Rev. 1994;58:755–805. doi: 10.1128/mr.58.4.755-805.1994. PubMed DOI PMC

Povedano-Priego C, Jroundi F, Lopez-Fernandez M, et al. Shifts in bentonite bacterial community and mineralogy in response to uranium and glycerol-2-phosphate exposure. Sci Total Environ. 2019;692:219–232. doi: 10.1016/j.scitotenv.2019.07.228. PubMed DOI

Quast C, Pruesse E, Yilmaz P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41:D590–D596. doi: 10.1093/nar/gks1219. PubMed DOI PMC

Setlow P. Spore resistance properties. Bact Spore. 2014 doi: 10.1128/microbiolspec. PubMed DOI

Shrestha R, Cerna K, Spanek R, et al. The effect of low-pH concrete on microbial community development in bentonite suspensions as a model for microbial activity prediction in future nuclear waste repository. Sci Total Environ. 2022 doi: 10.1016/j.scitotenv.2021.151861. PubMed DOI

Shrestha R, Černoušek T, Stoulil J, et al. Anaerobic microbial corrosion of carbon steel under conditions relevant for deep geological repository of nuclear waste. Sci Total Environ. 2021;800:149539. doi: 10.1016/j.scitotenv.2021.149539. PubMed DOI

Sirpa K, Annika H, Heino V. State-of-the-art study of foreign concepts of engineered elements in DGR. Praha, Czech Republic: SÚRAO; 2022.

SKB (2010) Buffer, backfill and closure process report for the safety assessment SR-Site. Svensk kärnbränslehantering (SKB)

Smart NR, Reddy B, Rance AP, et al. The anaerobic corrosion of carbon steel in saturated compacted bentonite in the Swiss repository concept. Corros Eng Sci Technol. 2017;52:113–126. doi: 10.1080/1478422X.2017.1316088. DOI

Stroes-Gascoyne S. Microbial occurrence in bentonite-based buffer, backfill and sealing materials from large-scale experiments at AECL’s Underground Research Laboratory. Appl Clay Sci. 2010;47:36–42. doi: 10.1016/j.clay.2008.07.022. DOI

Stroes-Gascoyne S, Sergeant C, Schippers A, et al. Biogeochemical processes in a clay formation in situ experiment: part D – microbial analyses – synthesis of results. Appl Geochem. 2011;26:980–989. doi: 10.1016/j.apgeochem.2011.03.007. DOI

Svensson D, Dueck A, Nilsson U, et al. Alternative buffer material. Status of the ongoing laboratory investigation of reference materials and test package 1. Stockholm, Sweden: Swedish Nuclear Fuel and Waste Management Co; 2011.

Trevors JT. DNA in soil: adsorption, genetic transformation, molecular evolution and genetic microchip. Antonie Van Leeuwenhoek. 1996;70:1–10. doi: 10.1007/bf00393564. PubMed DOI

Villar MV, Armand G, de Lesquen C, Herold P, Simo E, Mayor JC, Dizier A et al (2020) D7.1 HITEC. Initial state-of-the-art on THM behaviour of i) buffer clay materials and of ii) host clay materials. EURAD Project. Horizon 2020 No 847593

Wolf DC, Dao TH, Scott HD, Lavy TL. Influence of sterilization methods on selected soil microbiological, physical, and chemical properties. J Environ Qual. 1989;18:39–44. doi: 10.2134/jeq1989.00472425001800010007x. DOI

Comparing the effectiveness of different DNA extraction methods in MX-80 bentonite

Dramatic loss of microbial viability in bentonite exposed to heat and gamma radiation: implications for deep geological repository