Surface to Lower Biosphere Limit: Long-term Geobiology Reference Transect

1. Surface to Lower Biosphere Limit: Long-term Geobiology Reference Transect Why Biology Needs a DUSEL Duane P. Moser Desert Research Institute Las Vegas, NV

2. Outline: • Insights and frustrations from prior work • General concepts to incorporate into design • Specific ideas for long-term reference transect

3. Why Long-Term Reference Transect and why DUSEL? Learning from persistent challenges from past • Almost always sporadic samples of opportunity • Excavations always done for other purposes • Very limited capacity for repeat sampling

4. The Witwatersrand Deep Microbiology Project (1997-2003) TC Onstott and many, many others

5. Cren Group 2 "Subsurface" Group 2 Cren Group3 Cren Group 1c Cren Group 1b SAGMCG2 Marine Group 1 SAGMCG1 OPA2 Thermoprotei Archaeoglobi Methanococcales Thermococci OPA3/4 YNPFFA SAGMEG-1 Korarchaeota SAGMEG-2 Methanobacteria Bacteria Eukaroyotes FCG1 WSA2 pMG1 FCG2 FCG3 Halobacteria Northam Group 1 pMC2 Thermoplasma Methanomicrobia "Sed Archaea 1" 0.10 16S rRNA Tree by Thomas Gihring

7. Long-term Biosustainability in a High-energy, Low-diversity Crustal Biome Science: Accepted pending revisions L-H Lin, P-L Wang, D. Rumble, J. Lippmann-Pipke, E. Boice, L. Pratt, B. Sherwood Lollar, E. Brodie, T. Hazen, G. Andersen, T. DeSantis, D.P. Moser, D. Kershaw, and T.C. Onstott Brett Tipple, 3.3 kmbls in Mpneng

8. Why Long-Term ? Hole EB5 Evander Mine

9. Microbial Community Development in Boreholes service water drilling fluid borehole fluid, 1 hour borehole fluid, 48 hours borehole fluid, 30 days borehole fluid, 70 days unweighted arithmetic average clustering based on binary, presence/absence distance measures Bacterial 16S rDNA clone distribution a-Proteo. b-Proteo. g-Proteo. Nitrospira OP11 Firmicutes Bacteroidetes Synergistes 20 60 100 Percent of clones Borehole fluids, 30 days: Drilling fluid and service water communities no longer detected. Desulfotomaculum and taxa deeply-branched Firmicutes appear. Borehole fluid, 48 hours: Still primarily Proteobacteria Borehole fluid, 1 hour: Most similar to the drilling fluid community. Introduced community overprints indigenous community. Primarily Proteobacteria Drilling fluid Divergent from service water. Mostly Proteobacteria Comamonadaceae, Hydrogenophaga, Thiobacillus, Thauera, Pseudomonas, Acenitobacter, Alishewanella, etc. Borehole fluids, 70 days Population has stabilized. 7 taxa closely-related to Desulfotomaculum and deeply-branched Firmicutes. • Service water • Major source of introduced organisms. • Primarily Proteobacteria: • Comamonadaceae, Hydrogenophaga, Leptothrix, Alcaligenes, Nitrosomonas, Rhodobacter, etc.

10. image courtest of Gordon Southam South Africa Subsurface Firmicute Groups (SASFG) SASFG-6 SASFG-5 SASFG-4 SASFG-7 SASFG-3 SASFG-9 SASFG-8 * SASFG-1 SASFG-2 Major new bacterial lineages with one exception only found in South African subsurface below 1.5 km depth Complete genome for SASFG-1 (LBNL). Sulfate reducing, spore former, motile, nitrogen fixer. Tree by Thomas Gihring

11. Dec-98 Feb-99 Nov- 2001 Nov-2002 Stable (Indigenous?) Populations “SASFG-1” Isolate DR504 Bacterial T-RFLP data “community 16S rDNA fingerprint (3.2 kmbls Driefontein)”

12. Henderson Reference Transect • Stable, predictable, platform • Gold-standard reference site for • testing new technologies • Deep ecological reserve • Intact subsurface ecosystem • “Artificial fracture” • Track fluid movements (colonization history) • Repeated sampling

13. In situ Experiments: Artificial Fracture Zone? • Stevens and McKinley (H2 production in basalts) controversey… how important are fresh fracture surfaces and how fast do fault surfaces weather… do microbial communities respond to fault slip and other geological disturbances. • Seismicity: do biofilms lubricate faults? • Substrates (nutrient stimulation, recoverable mineral coupons)

14. Interface Between Oxic and Anoxic World

15. Logistics (Hardware) • Downhole packer • Multilevel sampler • U-tube with backfill • Valve at outlet Operation at ambient pressure? New systems from industry/DOE (e.g. oil, geothermal)?

16. Logistics (Materials) • Steel Casings/Valves • Corrosion = failure (stainless?) • Iron source = shifts in population • Hydrogen artifacts • Plastics/Rubber • PEEK, Delrin? (leaching?, degradation, pressure failure?) • Tubing (nylon, stainless)? • Titanium?

17. Logistics (Methods) • Distance • How far into the rock to escape mining influences? • Drilling/Coring • Drilling muds (e.g. chemicals, bentonite, introduced bugs) • Rotary drilling with airlift? • Grout • Legacy oxidation • Minerals oxidized during drilling • Steel cuttings remaining in hole

18. Biology DUSEL: Critical or Merely Important?

19. Conclusions • Henderson DUSEL a unique opportunity to finally do subsurface microbiology “right” • Long-term reference transect would be the gold-standard site for decades and adaptive to new technologies for life detection. • Different hydrology/lithology at Henderson expands subsurface biomes that will have been explored

21. Description of experiment: a controlled platform for long-term geobiology laboratory, offering near-continuous coverage of an intact subsurface ecosystem block from shallow-aquifer to near the lower biosphere limit. the tracking of fluid migration in three dimensions and the testing of hypotheses concerning deep microbial colonization history. deep ecological reserve and gold-standard reference site, which could be sampled repeatedly over decades in response to new technologies.

22. Description of experiment: Roughly ten side-wall boreholes of a minimum 500 m length ea. would be extended horizontally at interval, and into hotter depths by drilling into the mine floor. Holes would be sealed to ambient pressure and outfitted with sampling ports, packers and unreactive multilevel samplers to allow repeated sampling proximal to features and host rock types of interest. Holes in unsaturated zones would be sealed and packered to enable gas sampling and down-hole collection of surface biofilms. Microbial population structure in the boreholes would be assessed using the best available molecular tools, both temporally from time-zero and spatially to quantify the extent and persistence of mining-induced contamination. Facilities would be developed to enable to emplacement and recovery of long-term in situ mineral weathering and substrate addition experiments.

23. Anaerobic Ecosystems: Life’s Redox Footprint (What would you expect in the very deep subsurface?) O2 H20 + CO2 H2 concentration Aerobic Respiration 0 CH20(Burial) 0.05 nM Nitrate and Mn(IV) Respiration 0.2 nM Fe(III) Respiration 1-1.5 nM Sulfate Respiration Fermentations (release H2) 7-10 nM Methanogenesis/Acetogenesis (consume H2) 1) No available respiratory electron acceptors?

24. A. Endolithic Sulfate Reducers (a shot in the arm for radiolysis) A. Witwatersrand quartzite core from 1.95 km depth in fracture zone. Pink = rhodamine tracer. B. 35S auto-radiographic image of core. C. Sulfate reducing bacteria with AgS xtals in pore. C. B. Courtesy of Gordon Southam, Univ. of Western Ontario and Mark Davidson, Princeton University

25. Driefontein Consolidated Gold Mine

27. Methananobacterium • Actually an Archaeon (despite the name). • Makes Methane from CO or CO2 and H2 • Desulfotomaculum • Well known, sometimes thermophilic sulfate reducer • Uses acetate, H2, probably CO

28. D8A microbial population dsrA 16S rRNA mcrA

29. But wait a minute….. Methanogens and sulfate reducers are not supposed to cohabitate! 30 mM (radiolytic?) Sulfate Vast excess (20,000 - 200,000 X) of abiogenic H2 An perfectly-poised, electron acceptor-controlled system?

31. CONTRIBUTORS TC Onstott , Mark Davidson, Bianca Mislowack Princeton U Jim Fredrickson, Tom Gihring, and Fred Brockman PNNL Lisa Pratt, Eric Boice Indiana Univ. Barbara Sherwood Lollar, Julie Ward, Greg Slater U of Toronto Gordon Southam, Greg Wanger U of Western Ontario Ken Takai JAMSTEC Brett Baker UC Berkeley Tom Kieft New Mexico Tech Sue Pfiffner, Tommy Phelps U of Tennessee, ORNL Dave Boone, Adam Bonin, Anna Louise Reysenbach Portland State U Johanna Lippmann U of Potsdam Terry Hazen , Eoin Brodie, et al. LBNL Li-Hung Lin National Taiwan U Dawie Nel, Walter Seymor, Colin Ralston, etc. etc. Mine professionals Rob Wilson and staff Turgis Ltd. Consultants Derek Litterhauer and Esta VanHeerden Univ. of Free State Chrissie Rey, Faculty, students and staff U of Witwatersrand

32. The western Witwatersrand Basin Dolomite (Ca2+/Na+ ratio 2.4) 1 km Ventersdorp lava (Ca2+/Na+ ratio 1.4) 2 km 3 km 4 km 5 km Witwatersrand quartzite (Ca2+/Na+ ratio 0.12) 6 km d2H/d18O ratio and other chemistry matches other local waters aged to 3-30 MA Hydrogen isotope equilibration temp = 60.5 oC e.g. 3 - 5 km source depth 54 oC temp is higher than geothermal gradient would predict (upwelling) Ca2+/Na+ ratio and other geochem indicates water has not traversed shallower levels (lavas and dolomites) Thus water most likely aged meteoric, with long flow path, trapped in the Witwatersrand Supergoup (nearest outcrop = 11 km away.

33. 1) Boetius, A. 2005. Science, 307:1420-1422 2) Chapelle, F. H., . et al. 2002. Nature 415:312-315 3) Fry, N. K., J. K. Fredrickson, S. Fishbain, M. Wagner, and D. A. Stahl. 1997. Appl. Environ. Microbiol. 63:1498-1504. 4) Kelly, D.S. et al. 2005, Science,307: 1428-1434 5) Stevens, T. O., and J. P. Mckinley. 1995. Science 270:450-454 From Kelly, D.S. et al. 2005, Science, 1428-1434