Bentonite is an integral part of the engineered barrier system (EBS) in deep geological repositories (DGR) for nuclear waste, but its indigenous microorganisms may jeopardize long-term EBS integrity. To predict microbial activity in DGRs, it is essential to understand microbial reactions to the early hot phase of DGR evolution. Two bentonites (BCV and MX-80) with varied bentonite/water ratios and saturation levels (compacted to 1600 kg.m- 3 dry density/powder/suspension), were subjected to heat (90-150 °C) and irradiation (0.4 Gy.h- 1) in the long-term experiments (up to 18 months). Molecular-genetic, microscopic, and cultivation-based techniques assessed microbial survivability. Exposure to 90 °C and 150 °C notably diminished microbial viability, irrespective of bentonite form, with negligible impacts from irradiation or sample type compared to temperature. Bentonite powder samples exhibited microbial recovery after 90 °C heating for up to 6 months but not 12 months in most cases; exposure to 150 °C had an even stronger effect. Further long-term experiments at additional temperatures combined with the mathematical prediction of temperature evolution in DGR are recommended to validate the possible evolution and spatial distribution of microbially depleted zones in bentonite buffer around the waste canisters and refine predictions of microbial effects over time in the DGR.

Disposal Processes and Safety ÚJV Řež a s Hlavní 130 Husinec 250 68 Czech Republic

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

Radioactive Waste Repository Authority SÚRAO Dlážděná 6 Prague 11000 Czech Republic

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Aoki K, Sugita Y, Chijimatsu M, Tazaki K (2010) Impacts of thermo-hydro-mechanical experiments on the microbial activity in compacted bentonite at the Kamaishi Mine, Northeast Japan. Appl Clay Sci 47:147–15410.1016/j.clay.2008.12.016 DOI

Bartak D, Bedrníková E, Kašpar V et al (2023) Survivability and proliferation of microorganisms in bentonite with implication to radioactive waste geological disposal: strong effect of temperature and negligible effect of pressure. World J Microbiol Biotechnol 40:41. 10.1007/s11274-023-03849-0 10.1007/s11274-023-03849-0 PubMed DOI PMC

Beaton D, Pelletier P, Goulet RR (2019) Microbial Degradation of Cellulosic Material and Gas Generation: implications for the management of low- and Intermediate-Level Radioactive Waste. Front Microbiol 10:204. 10.3389/fmicb.2019.00204 10.3389/fmicb.2019.00204 PubMed DOI PMC

Bengtsson A, Pedersen K (2017) 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 137:203–21210.1016/j.clay.2016.12.024 DOI

Bengtsson A, Blom A, Johansson L et al (2017) Bacterial sulphide-producing activity in water saturated iron-rich Rokle and iron-poor Gaomiaozi bentonite at wet densities from 1750 to 1950 kg m – 3. Swedish Nuclear Fuel and Waste Management Co, Stockholm, Sweden

Bhana N, Zanwar AS, Trivedi V, Jain D (2013) A REVIEW: STEAM STERILIZATION A METHOD OF STERILIZATION. J Biol Amp Sci Opin 1:13810.7897/2321-6328.01222 DOI

Birgersson M, Hedström M, Karnland O, Sjöland A (2017) 12 - Bentonite buffer: Macroscopic performance from nanoscale properties. In: Apted MJ, Ahn J (eds) Geological Repository Systems for Safe Disposal of Spent Nuclear Fuels and Radioactive Waste (Second Edition). Woodhead Publishing, pp 319–364

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

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

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

Bruhn DF, Breckenridge CR, Tsang MN et al (1999) Irradiation of microbes from Spent Nuclear fuel Storage Pool environments. Idaho National Engineering and Environmental Lab

Burzan N, Murad Lima R, Frutschi M et al (2022) Growth and persistence of an aerobic Microbial Community in Wyoming Bentonite MX-80 despite anoxic in situ conditions. Front Microbiol 13 PubMed PMC

Butterworth SJ, Barton F, Lloyd JR (2023) Extremophilic microbial metabolism and radioactive waste disposal. Extremophiles 27:27. 10.1007/s00792-023-01312-4 10.1007/s00792-023-01312-4 PubMed DOI PMC

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

Červinka R, Vašíček R, Večerník P, Kašpar V (2018) SÚRAO technical report 419/2019: Kompletní charakterizace bentonitu BCV 2017

Chen S, Song L, Dong X (2006) Sporacetigenium mesophilum gen. nov., sp. nov., isolated from an anaerobic digester treating municipal solid waste and sewage. Int J Syst Evol Microbiol 56:721–725. 10.1099/ijs.0.63686-0 10.1099/ijs.0.63686-0 PubMed DOI

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

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

Daly MJ, Gaidamakova EK, Matrosova VY et al (2007) Protein oxidation implicated as the primary determinant of bacterial Radioresistance. PLOS Biol 5:e92. 10.1371/journal.pbio.0050092 10.1371/journal.pbio.0050092 PubMed DOI PMC

Davis NM, Proctor DM, Holmes SP et al (2018) Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. Microbiome 6:226. 10.1186/s40168-018-0605-2 10.1186/s40168-018-0605-2 PubMed DOI PMC

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

Du J, Gebicki JM (2004) Proteins are major initial cell targets of hydroxyl free radicals. Int J Biochem Cell Biol 36:2334–2343. 10.1016/j.biocel.2004.05.012 10.1016/j.biocel.2004.05.012 PubMed DOI

El Mendili Y, Abdelouas A, El Hajj H, Bardeau J-F (2013) Phase transitions of iron sulphides formed by steel microbial corrosion. Rsc Adv 3:26343–2635110.1039/c3ra45259j DOI

Engel K, Ford SE, Coyotzi S et al (2019) Stability of Microbial Community Profiles Associated with compacted Bentonite from the Grimsel Underground Research Laboratory. mSphere 4:e00601–e00619. 10.1128/mSphere.00601-19 10.1128/mSphere.00601-19 PubMed DOI PMC

Enning D, Garrelfs J (2014) Corrosion of Iron by sulfate-reducing Bacteria: new views of an old problem. Appl Environ Microbiol 80:1226–1236. 10.1128/AEM.02848-13 10.1128/AEM.02848-13 PubMed DOI PMC

Farda B, Djebaili R, Vaccarelli I et al (2022) Actinomycetes from caves: an overview of their diversity, Biotechnological Properties, and insights for their use in Soil environments. Microorganisms 10:453. 10.3390/microorganisms10020453 10.3390/microorganisms10020453 PubMed DOI PMC

Ghosal D, Omelchenko MV, Gaidamakova EK et al (2005) How radiation kills cells: survival of Deinococcus radiodurans and Shewanella oneidensis under oxidative stress. FEMS Microbiol Rev 29:361–375. 10.1016/j.fmrre.2004.12.007 10.1016/j.fmrre.2004.12.007 PubMed DOI

Gilmour KA, Davie CT, Gray N (2021) An indigenous iron-reducing microbial community from MX80 bentonite - A study in the framework of nuclear waste disposal. Appl Clay Sci 205:106039. 10.1016/j.clay.2021.10603910.1016/j.clay.2021.106039 DOI

Gilmour KA, Davie CT, Gray N (2022) 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 814:152660. 10.1016/j.scitotenv.2021.152660 10.1016/j.scitotenv.2021.152660 PubMed DOI

Gregory SP, Mackie JRM, Barnett MJ (2024) Radioactive waste microbiology: predicting microbial survival and activity in changing extreme environments. FEMS Microbiol Rev 48:fuae001. 10.1093/femsre/fuae001 10.1093/femsre/fuae001 PubMed DOI PMC

Hall DS, Behazin M, Jeffrey Binns W, Keech PG (2021) An evaluation of corrosion processes affecting copper-coated nuclear waste containers in a deep geological repository. Prog Mater Sci 118:100766. 10.1016/j.pmatsci.2020.10076610.1016/j.pmatsci.2020.100766 DOI

Hausmannová L, Dohnálková M, Matušková E et al (2023) Technical design of the deep geological repository 2023. SÚRAO

Havlová V, Prchal K, Hofmanová E (2020) Research support for the safety assessment of the deep geological repository final report. SÚRAO

Haynes HM, Pearce CI, Boothman C, Lloyd JR (2018) Response of bentonite microbial communities to stresses relevant to geodisposal of radioactive waste. Chem Geol 501:58–67. 10.1016/j.chemgeo.2018.10.00410.1016/j.chemgeo.2018.10.004 DOI

Hicks T, White M, Baldwin T et al (2009) Design options for the UK’s ILW geological disposal facility. Radioact Waste Manag 2009:11–15. 10.1115/ICEM2009-16241. Proc 2009 12th Int Conf Environ Remediat10.1115/ICEM2009-16241 DOI

Hlavackova V, Shrestha R, Hofmanova E et al (2023) A protocol for the extraction of viable bacteria for identification of bacterial communities in bentonite. Appl Clay Sci 232:106809. 10.1016/j.clay.2022.10680910.1016/j.clay.2022.106809 DOI

IAEA (2009) Classification of Radioactive Waste: General Safety Guide. IAEA, Austria, Vienna

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)

Kaminski A, Uhrynowska-Tyszkiewicz I, Stachowicz W (2021) Sterilisation by irradiation. In: Galea G, Turner M, Zahra S (eds) Essentials of tissue and cells banking. Springer International Publishing, Cham, pp 127–142

Kashefi K, Lovley DR (2003) Extending the Upper temperature limit for life. Science 301:934–934. 10.1126/science.1086823 10.1126/science.1086823 PubMed DOI

Kašpar V, Šachlová Š, 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/min1109096510.3390/min11090965 DOI

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

King F (2009) Microbiologically Influenced Corrosion of Nuclear Waste Containers. CORROSION 65:233–251. 10.5006/1.331913110.5006/1.3319131 DOI

King F, Hall DS, Keech PG (2017) Nature of the near-field environment in a deep geological repository and the implications for the corrosion behaviour of the container. Corros Eng Sci Technol 52:25–30. 10.1080/1478422X.2017.133073610.1080/1478422X.2017.1330736 DOI

Krisko A, Radman M (2010) Krisko A, Radman MProtein damage and death by radiation in Escherichia coli and Deinococcus radiodurans. Proc Natl Acad Sci USA 107:14373–14377. Proc Natl Acad Sci U S A 107:14373–7. 10.1073/pnas.1009312107 PubMed PMC

Kumpulainen S, Hagros A, Ville H (2022) State-of-the-art study of foreign concepts of engineered elements in DGR. SÚRAO, Praha, Czech Republic

Lee DW, Lee AH, Lee H et al (2017) Nocardioides litoris sp. nov., isolated from the Taean seashore. Int J Syst Evol Microbiol 67:2332–2336. 10.1099/ijsem.0.001954 10.1099/ijsem.0.001954 PubMed DOI

Liu H, Dang X, Zhang H et al (2019) Microbial diversity in bentonite, a potential buffer material for deep geological disposal of radioactive waste. IOP Conf Ser Earth Environ Sci 227:022010. 10.1088/1755-1315/227/2/02201010.1088/1755-1315/227/2/022010 DOI

Lopez-Fernandez M, Jroundi F, Ruiz-Fresneda MA, Merroun ML (2021a) Microbial interaction with and tolerance of radionuclides: underlying mechanisms and biotechnological applications. Microb Biotechnol 14:810–828. 10.1111/1751-7915.13718 10.1111/1751-7915.13718 PubMed DOI PMC

Lopez-Fernandez M, Matschiavelli N, Merroun ML (2021b) Chap. 7 - Bentonite geomicrobiology. In: Lloyd JR, Cherkouk A (eds) The Microbiology of Nuclear Waste Disposal. Elsevier, pp 137–155

Martinez-Moreno MF, Povedano-Priego C, Mumford AD et al (2024) Microbial responses to elevated temperature: evaluating bentonite mineralogy and copper canister corrosion within the long-term stability of deep geological repositories of nuclear waste. Sci Total Environ 915:170149. 10.1016/j.scitotenv.2024.170149 10.1016/j.scitotenv.2024.170149 PubMed DOI

Masurat P, Eriksson S, Pedersen K (2010a) 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 47:58–64. 10.1016/j.clay.2009.01.00410.1016/j.clay.2009.01.004 DOI

Masurat P, Eriksson S, Pedersen K (2010b) Evidence of indigenous sulphate-reducing bacteria in commercial Wyoming bentonite MX-80. Appl Clay Sci 47:51–57. 10.1016/j.clay.2008.07.00210.1016/j.clay.2008.07.002 DOI

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

Mattimore V, Battista JR (1996) Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation. J Bacteriol 178:633–637 10.1128/jb.178.3.633-637.1996 PubMed DOI PMC

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

Mijnendonckx K, Bleyen N, Van Gompel A et al (2022) pH and microbial community determine the denitrifying activity in the presence of nitrate-containing radioactive waste. Front Microbiol 13 PubMed PMC

Morozkina EV, Slutskaya ES, Fedorova TV et al (2010) Extremophilic microorganisms: biochemical adaptation and biotechnological application (review). Appl Biochem Microbiol 46:1–14. 10.1134/S000368381001001110.1134/S0003683810010011 PubMed DOI

Motamedi M, Karland O, Pedersen K (1996) Survival of sulfate reducing bacteria at different water activities in compacted bentonite. FEMS Microbiol Lett 141:83–87. 10.1111/j.1574-6968.1996.tb08367.x10.1111/j.1574-6968.1996.tb08367.x DOI

Mulligan CN, Yong RN, Fukue M (2009) Some effects of microbial activity on the evolution of clay-based buffer properties in underground repositories. Appl Clay Sci 42:331–335. 10.1016/j.clay.2008.03.00210.1016/j.clay.2008.03.002 DOI

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

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

OECD, Nuclear Energy A (2008) Radioactive Waste Management and Decommissioning. Nuclear Energy Outlook 2008. OECD, pp 240–270

OECD, Nuclear Energy Agency (2003) Engineered Barrier systems and the Safety of Deep Geological repositories: State-of-the-art report. OECD

OECD, Nuclear Energy Agency (2006) Radioactive Waste Management Programmes in OECD/NEA Member Countries. OECD

Park S-Y, Zhang Y, O’Loughlin EJ et al (2024) Temperature-dependent microbial reactions by indigenous microbes in bentonite under fe(III)- and sulfate-reducing conditions. J Hazard Mater 465:133318. 10.1016/j.jhazmat.2023.133318 10.1016/j.jhazmat.2023.133318 PubMed DOI

Pedersen K (2000) Microbial processes in radioactive waste disposal. Sweden

Pedersen K (2010) Analysis of copper corrosion in compacted bentonite clay as a function of clay density and growth conditions for sulfate-reducing bacteria. J Appl Microbiol 108:1094–1104. 10.1111/j.1365-2672.2009.04629.x 10.1111/j.1365-2672.2009.04629.x PubMed DOI

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

Pospiskova I, Dobrev D, Kouril M et al (2017) Czech national programme and disposal canister concept. Corros Eng Sci Technol 52:6–10. 10.1080/1478422X.2017.1300379

Povedano-Priego C, Jroundi F, Solari PL et al (2023) Unlocking the bentonite microbial diversity and its implications in selenium bioreduction and biotransformation: advances in deep geological repositories. J Hazard Mater 445:130557. 10.1016/j.jhazmat.2022.130557 10.1016/j.jhazmat.2022.130557 PubMed DOI

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

Ratto M, Itavaara M (2012) Microbial activity in bentonite buffers. Literature study. VTT Technical Research Centre of Finland

Reisz JA, Bansal N, Qian J et al (2014) Effects of Ionizing Radiation on Biological molecules—mechanisms of damage and emerging methods of detection. Antioxid Redox Signal 21:260–292. 10.1089/ars.2013.5489 10.1089/ars.2013.5489 PubMed DOI PMC

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

Shrestha R, Cerna K, Spanek R et al (2022) 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 808:151861. 10.1016/j.scitotenv.2021.151861 10.1016/j.scitotenv.2021.151861 PubMed DOI

SKB (2010a) Data report for the safety assessment SR-Site. Svensk kärnbränslehantering (SKB)

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

Staley JT, Irgens RL, Brenner DJ (1987) Enhydrobacter aerosaccus gen. nov., sp. nov., a Gas-Vacuolated, facultatively anaerobic, Heterotrophic Rod. Int J Syst Evol Microbiol 37:289–291. 10.1099/00207713-37-3-28910.1099/00207713-37-3-289 DOI

Stroes-Gascoyne S (2010) Microbial occurrence in bentonite-based buffer, backfill and sealing materials from large-scale experiments at AECL’s Underground Research Laboratory. Appl Clay Sci 47:36–42. 10.1016/j.clay.2008.07.02210.1016/j.clay.2008.07.022 DOI

Stroes-Gascoyne S, West JM (1997) Microbial studies in the Canadian nuclear fuel waste management program. FEMS Microbiol Rev 20:573–590. 10.1111/j.1574-6976.1997.tb00339.x 10.1111/j.1574-6976.1997.tb00339.x PubMed DOI

Stroes-Gascoyne S, Lucht LM, Borsa J et al (1995) Radiation Resistance of the natural Microbial Population in buffer materials. MRS Online Proc Libr Arch 353. 10.1557/PROC-353-345

Stroes-Gascoyne S, Hamon CJ, Vilks P, Gierszewski P (2002) Microbial, redox and organic characteristics of compacted clay-based buffer after 6.5 years of burial at AECL’s Underground Research Laboratory. Appl Geochem 17:1287–1303. 10.1016/S0883-2927(02)00020-310.1016/S0883-2927(02)00020-3 DOI

Svoboda J, Šťástka J, Vašíček R et al (2022) Design of the Czech concept for the buffer, backfill, plugs, and sealing of disposal chambers for other RAW; the sealing of other underground spaces and construction elements. SÚRAO

Timkina E, Drábová L, Palyzová A et al (2022) Kocuria strains from Unique Radon Spring Water from Jachymov Spa. Fermentation 8:35. 10.3390/fermentation801003510.3390/fermentation8010035 DOI

Todoriki S, Furuta M, Nagai T, Hayashi T (2000) Modification of radiation resistance of Bacillus spores by water. Radiat Phys Chem 57:531–534. 10.1016/S0969-806X(99)00420-X10.1016/S0969-806X(99)00420-X DOI

Urios L, Marsal F, Pellegrini D, Magot M (2013) Microbial Diversity at Iron-Clay interfaces after 10 years of Interaction inside a deep Argillite Geological formation (Tournemire, France). Geomicrobiol J 30:442–453. 10.1080/01490451.2012.70522710.1080/01490451.2012.705227 DOI

Vachon MA, Engel K, Beaver RC et al (2021) Fifteen shades of clay: distinct microbial community profiles obtained from bentonite samples by cultivation and direct nucleic acid extraction. Sci Rep 11:22349. 10.1038/s41598-021-01072-1 10.1038/s41598-021-01072-1 PubMed DOI PMC

Yu H-Y, Wang Y, Chen P et al (2014) The effect of ammonium chloride and urea application on soil bacterial communities closely related to the reductive transformation of pentachlorophenol. J Hazard Mater 272:10–19. 10.1016/j.jhazmat.2014.02.037 10.1016/j.jhazmat.2014.02.037 PubMed DOI