1. Surface to Lower Biosphere Limit: Long-term Geobiology Reference Transect Why Biology Needs a DUSEL Duane P. Moser 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? Almost always sporadic samples of opportunity Excavations always done for other purposes Very limited capacity for repeat sampling Learning from persistent challenges from past

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

5. SAGMCG1 SAGMCG2 Cren Group 1b Cren Group 1c Cren Group 2 "Subsurface" Group 2 Cren Group3 Korarchaeota YNPFFA OPA3/4 Thermoprotei OPA2 Methanomicrobia Eukaroyotes Bacteria Northam Group 1 WSA2 Thermoplasma Halobacteria pMG1 FCG3 Methanobacteria pMC2 "Sed Archaea 1" FCG1 SAGMEG-1 SAGMEG-2 Archaeoglobi FCG2 Methanococcales Thermococci 0.10 Marine Group 1 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. Hole EB5 Evander Mine Why Long-Term ?

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

10. South Africa Subsurface Firmicute Groups (SASFG) * SASFG-1 SASFG-2 SASFG-3 SASFG-4 SASFG-5 SASFG-6 SASFG-7 SASFG-9 SASFG-8 image courtest of Gordon Southam 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. Stable (Indigenous?) Populations Dec-98 Feb-99 Nov- 2001 Nov-2002 Bacterial T-RFLP data “community 16S rDNA fingerprint (3.2 kmbls Driefontein)” Isolate DR504 “SASFG-1”

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

16. 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? Logistics (Materials)

17. 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 Logistics (Methods)

18. Biology DUSEL: Critical or Merely Important?

19. 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 Conclusions

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?) Methanogenesis/Acetogenesis (consume H 2 ) H 2 0 + CO 2 Aerobic Respiration Nitrate and Mn(IV) Respiration Fermentations (release H 2 ) CH 2 0 (Burial) O2O2 Sulfate Respiration 1-1.5 nM 0 0.05 nM H 2 concentration Fe(III) Respiration 7-10 nM 0.2 nM 1) No available respiratory electron acceptors?

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

25. Driefontein Consolidated Gold Mine

27. -Methananobacterium -Actually an Archaeon (despite the name). -Makes Methane from CO or CO 2 and H 2 -Desulfotomaculum -Well known, sometimes thermophilic sulfate reducer -Uses acetate, H2, probably CO

28. D8A microbial population 16S rRNA dsrA mcrA

29. But wait a minute….. Methanogens and sulfate reducers are not supposed to cohabitate! 30  M (radiolytic?) Sulfate Vast excess (20,000 - 200,000 X) of abiogenic H 2 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 ReysenbachPortland 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 VanHeerdenUniv. of Free State Chrissie Rey, Faculty, students and staffU of Witwatersrand

32.  2 H/  18 O ratio and other chemistry matches other local waters aged to 3-30 MA Hydrogen isotope equilibration temp = 60.5 o C e.g. 3 - 5 km source depth Ca 2+ /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. The western Witwatersrand Basin Ventersdorp lava (Ca 2+ /Na + ratio 1.4 ) Witwatersrand quartzite (Ca 2+ /Na + ratio 0.12 ) Dolomite (Ca 2+ /Na + ratio 2.4 ) 1 km 2 km 3 km 4 km 5 km 6 km 54 o C temp is higher than geothermal gradient would predict (upwelling)

33. From Kelly, D.S. et al. 2005, Science, 1428-1434 Lost City (1, 3)Lidy Spring (2) Columbia R. Basalt (3, 5) D8A (this work) MarineContinental RockperidotiteBasalt quartzite pH10 -116.97.5 - 9.59.1 CO 2 low55 mM1 - 3 mMlow Temp60 - 75 o C59 o C18 - 20 o C54 - 61 o C CH 4 1 - 2 mM0.1 mM 2 - 209  M 17.5 mM H2H2 <1 to 15 mM0.0013 mM0 - ca. 80 mM0.165 mM sulfate1 - 4 mM1.3 mM0.004 - 1.4 mM0.03 mM DominatedMethanogen SRB (firmicutes) MethanogenAcetogen? Methanogen Various SRBs firmicutes Methanogen SRB (firmicutes) 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