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Technical Report 97-22 SE9800189 TECHNICAL REPORT 97-22 Investigations of subterranean micro- organisms and their importance for performance assessment of radioactive waste disposal Results and conclusions achieved during the period 1995 to 1997 Karsten Pedersen Goteborg University, Institute of Cell and Molecular Biology, Department of General and Marine Microbiology, Goteborg, Sweden November 1997 SVENSK KARNBRANSLEHANTERING AB SWEDISH NUCLEAR FUEL AND WASTE MANAGEMENT CO P.O.BOX 5864 S-102 40 STOCKHOLM SWEDEN PHONE +46 8 459 84 00 FAX+46 8 661 57 19 INVESTIGATIONS OF SUBTERRANEAN MICROORGANISMS AND THEIR IMPORTANCE FOR PERFORMANCE ASSESSMENT OF RADIOACTIVE WASTE DISPOSAL RESULTS AND CONCLUSIONS ACHIEVED DURING THE PERIOD 1995 TO 1997 Karsten Pedersen Goteborg University, Institute of Cell and Molecular Biology, Department of General and Marine Microbiology, Goteborg, Sweden November 1997 This report concerns a study which was conducted for SKB. The conclusions and viewpoints presented in the report are those of the author(s) and do not necessarily coincide with those of the client. Information on SKB technical reports from 1977-1978 (TR 121), 1979 (TR 79-28), 1980 (TR 80-26), 1981 (TR 81-17), 1982 (TR 82-28), 1983 (TR 83-77), 1984 (TR 85-01), 1985 (TR 85-20), 1986 (TR 86-31), 1987 (TR 87-33), 1988 (TR 88-32), 1989 (TR 89-40), 1990 (TR 90-46), 1991 (TR 91-64), 1992 (TR 92-46), 1993 (TR 93-34), 1994 (TR 94-33), 1995 (TR 95-37) and 1996 (TR 96-25) is available through SKB. INVESTIGATIONS OF SUBTERRANEAN MICROORGANISMS AND THEIR IMPORTANCE FOR PERFORMANCE ASSESSMENT OF RADIOACTIVE WASTE DISPOSAL RESULTS AND CONCLUSIONS ACHIEVED DURING THE PERIOD 1995 TO 1997 Karsten Pedersen Goteborg University, Institute of Cell and Molecular Biology, Department of General and Marine Microbiology, Medicinaregatan 9C, SE-413 90 Goteborg, Sweden, Telephone: +46 31 773 25 78, Telefax +46 31 773 25 99, e-mail: [email protected] November 1997 NEXT PAGE(S) left BLANK ABSTRACT In 1987, microbiology became a part of the Swedish scientific program for the safe disposal of high level nuclear waste (HLW). The goal of the microbiology sub-program is to understand how subterranean microorganisms will interact with the performance of a future HLW repository. The Swedish research program on subterranean microbiology has mainly been performed at two sites in granitic rock aquifers at depths ranging from 70 m down to 1240 m; the Stripa research mine in the middle of Sweden and the Aspo hard rock laboratory (HRL) situated on the south eastern coast of Sweden. Some work has also been performed in co- operation with other national or international research groups in Sweden Canada and at the natural analogue sites in Oklo in Gabon and Maqarin in Jordan. The most recent report in the SKB technical report series on microbiology and performance assessment, SKB TR 95-10, gave the state of the art regarding microorganisms and their importance for performance assessment. That report is recommended as a source of knowledge about basic microbiology, microbial ecology of subterranean environments and the nuclear waste disposal concept in a microbiological perspective. The present report summarises results and conclusions achieved during the period 1995 to 1997 and is a continuation of SKB TR 95-10. The report is structured as summary which explains and analyses the obtained results and conclusions in a performance assessment perspective. The scientific basis for the summary is an enclosed series of eleven papers of which eight have gone through an international peer review process for publication in international scientific journalsand reports and papers published earlier NEXT PAQE(S) left BLANK SAMMANFATTNING Mikrobiologiska undersökningar blev 1987 en del av det svenska forskningsprogrammet för slutförvar av högaktivt, använt kärnbränsle. Målet för detta delprogram är att förstå hur mikroorganismer i och kring ett förvar kan interagera med funktionerna hos ett framtida förvar. Forskning inom det mikrobiologiska programmet har huvudsakligen utförts på två platser i granitiskt berg på djup mellan 70 och 1240 m; Stripa forskningsgruva i Bergslagen och Äspö berglaboratorium strax utanför Oskarshamn. En del forskningsarbete har också utförts i samarbete med andra nationella och internationella forskningsgrupper i Sverige, Kanada och vid den naturliga analogierna Oklo i Gabon och Maqarin i Jordanien. Den senast utgivna tekniska rapporten om mikrobiologisk forskning i SKB:s rapport serie, SKB TR 95-10, redogjorde ingående för kunskapsläget om mikroorganismer och deras betydelse för utvärdering av olika förvarsfunktioner. Den rapporten rekommenderas som en kunskapsbas om grundläggande mikrobiologi, den djupa biosfärens ekologi och slutförvarskonceptet i ett mikrobiologiskt perspektiv. Den nu publicerade rapporten summerar resultat och konklusioner som har uppnåtts under perioden 1995 till 1997 och utgör en fortsättning på SKB TR 95-10. Rapporten är strukturerad som en sammanfattning vilken förklarar och analyserar uppnådda resultat och konklusioner ur ett förvarsperspektiv. Den vetenskapliga basen för rapporten utgörs av elva bifogade artiklar av vilka 8 har genomgått granskning av internationella experter före publicering i internationella vetenskapliga tidskrifter samt rapporter och artiklar publicerade tidigare. NEXT PAOE(S) left BLANK CONTENTS List of papers ix List of appendix xv Summary and conclusions xvii 1 Introduction 1 2 Research tasks 5 3 Currently achieved results and research in progress 7 3.1 The subterranean biosphere 7 3.1.1 Evidence for the existence of a deep subterranean biosphere 7 3.1.2 Subterranean microbes and biogeochemicalprocesses 5 3.1.3 The hydrogen dependent subterranean biosphere hypothesis 9 3.1.4 The deep hydrogen driven biosphere 14 3.1.5 Research task, conclusions and performance assessment relation 15 3.2 Microbial production and consumption of gases 16 3.2.1 Dissolved gas in groundwater 16 3.2.2 Research task, conclusions and performance assessment relation 20 3.3 Microbial reducing activity 20 3.3.1 Research task, conclusions and performance assessment relation 22 3.4 Microbial corrosion of copper 23 3.4.1 Research task, conclusions and performance assessment relation 25 3.5 Microbial recombination of radiolysis products 26 3.5.1 Research task, conclusions and performance assessment relation 26 3.6 Microbial influence on radionuclide migration 26 3.6.1 Research task, conclusions and performance assessment relation 27 3.7 Alkaliphilic microbes and concrete 28 3.7.1 Research task, conclusions and performance assessment relation 29 4 References 29 vii NEXT PAQE(S) left BLANK LIST OF PAPERS PAPER 1 Investigation of the potential for microbial contamination of deep granitic aquifers during drilling using 16S rRNA gene sequencing and culturing methods 33 1 Abstract 35 2 Introduction 35 3 Materials and methods 36 3.1 Description of the SELECT site and drilling procedures 36 3.2 Sampling procedures 38 3.3 Groundwater chemistry 39 3.4 Determination of the total number of bacteria 39 3.5 Viable count and MPN of sulphate reducing bacteria 39 3.6 DNA extraction, PCR-amplification, cloning and sequencing of the 16S-rRNAgene 40 3.7 Sequence analysis 41 4 Results 41 4.1 Groundwater chemistry 41 4.2 Total number of bacteria 42 4.3 Viable counts and MPN of SRB 44 4.4 16S rRNA gene distribution 44 4.5 Accession numbers 46 5 Discussion 46 6 Conclusions 51 7 Acknowlesgments 51 8 References 52 PAPER 2 Evidence of ancient life at 207 m depth in a granitic aquifer 55 1 Abstract 57 2 Introduction 57 3 Materials and methods 58 3.1 Stable isotope analyis 58 3.2 Transmission electron microscopy 59 3.3 Scanning electron microscopy 59 4 Results and discussion 59 5 Acknowledgements 65 6 References 65 PAPER 3 16S rRNA gene diversity of attached and unattached bacteria in boreholes along the access tunnel to the Aspo hard rock laboratory, Sweden 67 1 Abstract 69 2 Introduction 69 IX 3 Materials and methods 70 3.1 Description of study site and sampling procedures 70 3.2 Groundwater chemistry 72 3.3 Attachment and growth of bacteria 72 3.4 Scanning electron microscopy 72 3.5 Determination of the total number of bacteria 72 3.6 Enrichment and isolation of IRB and SRB and viable count of SRB 73 3.7 DNA extraction, PCR-amplification, cloning and sequencing of the 16SrRNAgene 73 3.8 Sequencing of the 16S rRNA genes from cultures 74 3.9 Sequence analysis 75 4 Results 75 4.1 Groundwater composition and number of bacteria 75 4.2 SEM of attached bacteria 76 4.3 16S rRNA gene diversity of attached and unattached bacteria along the tunnel 78 4.4 16S rRNA gene diversity of isolated SRB and IRB 80 4.5 The correlation between flow and 16S rRNA gene distribution in theRZ fracture zone 82 4.6 Accession numbers 83 5 Discussion 83 5.1 Groundwater composition and bacteria present 83 5.2 16S rRNA gene diversity and distribution of unattached and attached bacteria in Aspo HRL 85 5.3 Identity of the sequences found 87 5.4 16S rRNA gene sequences as tracers for groundwater mixing 88 6 Acknowledgments 89 7 References 89 PAPER 4 RNA extraction from deep granitic groundwaters at Aspo and hybridization with group-specific 16S rRNA-directed oligonucleotide probes 93 1 Abstract 95 2 Introduction 95 3 Materials and methods 96 3.1 Study site 96 3.2 Sampling of groundwater microorganisms for RNA extraction 96 3.3 RNA extraction 97 3.4 RNA quantification 98 3.5 RNA slot blots 98 3.6 Oligonucleotide probes and specificity control 98 3.7 Slot blot hybridization with [gamma-32P]-ATP-labelled probes 99 3.8 Enrichment cultures for Acinetobacter 99 4 Results 100 4.1 RNA extraction and purification of deep groundwater samples 100 4.2 Probe detection limits 104 4.3 Acinetobacter enrichments 105 5 Discussion 106 5.1 RNA extraction and purification 106 5.2 Detection limits in hybridization with deep groundwater 16S rRNA..
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