1. Distribution and activity of sulphate-reducing bacteria in Äspö groundwater Karsten Pedersen, Chalmers

2. Why sulphate-reducing bacteria? Equipment and methods Sulphide production rates Two cases in the Äspö tunnel On-going research Outline

3. Why sulphate-reducing bacteria? A copper mini-canister that has been exposed to vivid microbial sulphide formation from sulphate, possibly with H 2 from the corroding cast iron insert as the electron donor. Image from: Smart, N. Rance, A. Reddy, B. Fennell, P. Winsley, R. 2012. Analysis of SKB MiniCan Experiment 3. SKB Technical Report TR-12-09

5. Main site in the ÄSPÖ tunnel The MICROBE laboratory at 450 m depth Packer systems 5

6. Flow cells for attachment and growth of microorganisms The flow cell with flat surfaces The flow cell with crushed rock

7. Stainless steel circulations with crushed rock flow cells and E h electrodes at 450 m depth and a pressure range between 2 – 3.5 MPa 7

8. Sulphide formation from H 2 is fast at low H 2 concentrations 1.1 ×10  15 mol S 2  h  1 cell  1 Pedersen, K. 2012, Subterranean microbial populations metabolize hydrogen and acetate under in situ conditions in granitic groundwater at 450 m depth in the Äspö Hard Rock Laboratory, Sweden, FEMS Microbiology Ecology, 81 217-229. Pedersen, K. 2012, Influence of H 2 and O 2 on sulphate-reducing activity of a subterranean community and the coupled response in redox potential, FEMS Microbiology Ecology, 82 653-665.

9. Hydrogen in groundwater

10. Phages are abundant in Äspö groundwater Large diversity and phage / bacteria ratios ranging from 1.1. to 18; average was 12 Kyle J.E., Eydal H.S.C., Ferris F.G. and Pedersen K. (2008) Viruses in granitic groundwater from 69 to 450 m depth of the Äspö hard rock laboratory, Sweden. The ISME Journal 2, 571-574. Isolated phage specific for Desulfovibrio aespoeensis Eydal H.S.C., Jägevall S., Hermansson M. and Pedersen K. (2009) Bacteriophage lytic to Desulfovibrio aespoeensis isolated from deep groundwater. The ISME Journal 3, 1139-1147. 10

11. The viral shunt and the total number of cells (TNC) 11 virus microorganisms Cell debris Cells

12. Rate per cell: 1.1×10  15 mol S 2  h  1 cell  1 Maximal number of sulphate-reducing bacteria: 10 6 cells mL  1 1.1 ×10  6 mol S 2  L  1 h  1 2.6 ×10  5 mol S 2  L  1 day  1 9.4 ×10  3 mol S 2  L  1 year  1 1000 mol S 2  L  1 100 000 years  1 TOO MUCH FOR A REPOSITORY!! Maximal, theoretical sulphide production in groundwater

13. KJ0052F01, KJ0052F03 and KJ0050F01 at MICROBE 450 m depth KA3110A (400 m) and KA3385 (420 m) Two cases in the Äspö tunnel KJ0052F01 KJ0052F03 KJ0050F01 KA3110A KA3385A

14. KJ0052F01, KJ0052F03 and KJ0050F01 at MICROBE KJ0050F01KJ0052F01 KJ0050F03 Analysiscells mL -1 cells mL -1 cells mL -1 Total number of cells45 000170 0009 500 Sulphate-reducing bacteria28030 00030 14 Hallbeck, L. and Pedersen, K. 2008, Characterization of microbial processes in deep aquifers of the Fennoscandian Shield, Applied Geochemistry, 23 1796-1819.

15. Long-term observations Sulphate-reducing bacteriaSulphate Drilling of KA3386A drained >15 000 m 3 from the MICROBE aquifers

16. The KA3386A drainage induced vivid sulpide production from H 2

17. KA3110A (400 m) KA3385 (420 m) KA3110A KA3385A

18. 454 Pyrosequencing results for KA3110A 10.4 % sulphate-reducing bacteria

19. 454 Pyrosequencing results for KA3385A 30.7 % sulphate-reducing bacteria

20. Presence in both groundwater types H 2 related sulphide producing activity? KA3110A KA3385A

21. On-going……. And: work with DNA analysis of Biofilms from flow cells is in progress

22. Groundwater charaterization using DNA signatures……

23. H 2 is a key player in the deep biosphere – tested and supported by data. CH 4 may as well be important, but K m for anaerobic oxidation of CH 4 is 10 000 times higher than K m for H 2 and therefore often very slow. High pressure experiments needed - can be tested with in situ experiments at Äspö. DNA diversity profiles reveal groundwater origin and mixing – collect data and compare with existing Äspö models. Hypotheses

24. Acknowledgements MICROBIAL ANALYTICS SWEDEN AB  Johanna Arlinger, Andreas Bengtsson, Ulrika Björkner, Alexandra Chukharkina, Johanna Edlund, Lena Eriksson, Anna Hallbeck, Björn Hallbeck, Maria Hallbeck, Lotta Hallbeck, Jessica Johansson, Linda Johansson, Sara Jägevall, Sara Lydmark, Anna Pääjärvi and Lisa Rabe. Personnel at Äspö and ONKALO underground research laboratories and numerous colleagues and co-authors over 25 years of deep biosphere exploration THE RESEARCH LEADING TO THESE RESULTS WAS FUNDED BY  Census of Deep Life (CoDL), Carnegie Institution of Washington - Deep Carbon Observatory, USA  European Union’s European Atomic Energy Community’s (Euratom) Seventh Framework Programme FP7/2007-2011 under grant agreement no. 212287 (RECOSY project).  NOVA Oskarshamn  Posiva Oy, Olkiluoto, Finland  The Swedish Nuclear Fuel and Waste Management Co.  The Swedish Research Council 24

25. Thank you for your attention