1. Life in the Slow Lane: Deep subsurface geomicrobiology Tom Kieft New Mexico Tech

2. Outline Subsurface geomicrobiology -- background Witwatersrand Deep Microbiology Project Deep Underground Science and Engineering Laboratory (DUSEL) Geomicrobiology Low-level counting applications

3. Life in the Subsurface is Microbial Microorganisms –Bacteria –Archaea –Protozoa –Fungi –viruses

4. Why study subsurface microbes? Reveal unknown metabolic capabilities and ecosystems Applications of novel microbes Bioremediation of contaminated aquifers Understanding waste repositories Analogs for life on other planets?

5. macroorganisms microorganisms

6. Microbes couple oxidation of fuels (electron donors) with reduction of oxidants (electron acceptors). Subsurface Fuels: Microbes near the surface depend on photosynthetically generated organic carbon. The deep biosphere may depend on geochemically derived energy sources: H 2, CH 4, etc. or < 20 kJ/mole

7. Subsurface microbiology 1986-present Drilling and tracer technologies Extended known biosphere to >3 km Revealed biomass & biodiversity Isolates in culture collections Linked microbial activity with geological interfaces Slow rates of subsurface microbial activity Indications of autotrophic ecosystems

8. SubsurfaceMicrobialBiogeochemicalCycling Sampling Microscopy & Mineral Geochemistry AqueousGeochemistry Environment Dissolved Gases, Cosmogenic & Stable Isotopes & Stable Isotopes Membrane lipids Isolates & Archives Function 16Sr DNA Community Structure Enrichments genes, & enzymes

9. Subsurface biomass was considered insignificant but is now recognized as a major fraction of planetary biomass (greater than surface biomass?) Subsurface microbial populations are: diverse, active, unusual, possess novel traits, represent an exploitable resource

10. EarthLab

11. Life in the slow lane!

13. Witwatersrand Deep Microbiology Project Team T.C. Onstott - Princeton Duane Moser, Tom Gihring, Jim Fredrickson - PNNL Barbara Sherwood Lollar - Univ. of Toronto Lisa Pratt - Indiana Univ. Tom Kieft – New Mexico Tech Susan Pfiffner - Univ. Tenn. Tommy Phelps - ORNL David Boone - Portland State Univ. David Balkwill - Florida State University Gordon Southam - Univ. Western Ontario Johanna Lippmann - Lamont-Doherty Earth Observatory Ken Takai - JAMSTEC Esta van Heerden, Derek Litthaur -Univ. Free State David Boone - Portland State Univ. David Balkwill - Florida State University Gordon Southam - Univ. Western Ontario Johanna Lippmann - Geoforschungzentrum Ken Takai - JAMSTEC Esta van Heerden, Derek Litthaur -Univ. Free State Many others, especially students.

14. Deep, sequestered microbial communities? Evander 3.0 Ga basement 2.0 Ga meteorite impact Uplift ~2 km at 90 myr

15. 2.0 Ga 2.9 Ga 2.7 Ga 2.0 Ga300 Ma 2.3 Ga Basement 3.4 Ga 9-15 o C/km 25 o C/km 20 o C/km 120 o C Geothermal gradients:

16. Geomicrobiological sampling Rocks from freshly mined surfaces Fissure water from flowing boreholes –Filtered to concentrate cells including massive filtering (~10,000 liters) –In situ enrichment devices Cores -- especially useful for sampling rock matrix, fractures Biofilms

17. Gas analysis of fissure waters CH 4 (30-80%) C 2 + (3-4%) H 2 (up to 30%) He (up to 10%) balance N 2 some NH 3 ?

18. Geological Cross Section from West Driefontein to East Driefontein Service water is then pumped 1-2 kilometers to the stopes Where the carbon leader is mined To cool circulating air, control dust levels, and cool drilling equipment From the stopes, the now hot water flows to the base of the shaft and is pumped to the surface (red arrows) Where it is chilled, treated and recirculated to the subsurface Dolomite water drawn from the IPC pump chamber at shaft #4 augments supply Service water is chilled to 4°C and treated with chlorine and bromine before it descends shaft #5 to mining levels (blue arrows)Service water is chilled to 4°C and treated with chlorine and bromine before it descends shaft #5 to mining levels (blue arrows)

19. Service, dolomite, and fissure waters: pe vs. pH

20. Mean Precipitation Ancient Water Deep Hydro- thermal Water? Service Water LMWL Hot Springs

21. * SASFiG-9 (isolated) Detected within a water-bearing dyke/fracture at 3.2 Km depth. strictly anaerobic; iron-reducer optimal growth temperature = 60 o C virgin rock temp = ~ 45 o C * SASFiG-1 SASFiG-2 SASFiG-3 SASFiG-4 SASFiG-5 SASFiG-6 SASFiG-7 SASFiG-9 SASFiG-8 * image courtesy of Gordon Southam Culture-dependent and culture-independent geomicrobiological characterization: Novel indigenous microbes and communities Novel and unusual deeply branched sequences may be indicative of ancestral linkages, (early life?), Novel products for biomed and biotech applications Novel Bacterial lineages unique to the SA deep- subsurface: South Africa Subsurface Firmicutes Groups (SASFiG) 1  m

22. Geomicrobiology at DUSEL Probe lower limit of the biosphere. Test “geogas” hypothesis: ecosystems dependent on geochemically generated H 2. Study adaptations for long-term persistence of microbial communities Geologic interfaces

23. Why we need DUSEL for geomicrobiology: Need for a dedicated site, with continuous long- term access, infrastructure, etc. Access to great depth (>3 km) –Test limits of life, depth of biosphere –Ecosystems based on H 2, “geogas” Monitor human impacts on the subsurface Biotechnical applications –In situ mining –Bioremediation –Novel enzymes, pharmaceuticals, etc.

24. What are the big research questions? What energy and carbon sources are available in the deep subsurface? Importance of “Geogas”? What are the sources of H 2 ? Rates of H 2 generation? Independence from photosynthesis? Are these ecosystems suitable analogs for possible subsurface life on other planets? Are there subsurface microbes and communities that are selected for and adapted to the extreme conditions of the subsurface? How has the metabolism of indigenous communities influenced subsurface geochemistry?

25. More research questions: What are the in situ rates of metabolism? What adaptations do microbes have that enable persistence for geologic time periods under extreme conditions? –Low nutrient flux, high temperature, extreme pH, high pressure, etc. How do subsurface microbes maintain/repair macromolecular structures? Do subsurface microbes represent early life on earth?

26. Technical requirements, desires, etc. Access to multiple locations with varied geology (could be single or multiple sites) –Igneous, metamorphic, and sedimentary rocks Access to locations with geological interfaces, geochemical gradients Access to pristine “green fields” (unmined, unimpacted by mining) Access at multiple depths Access to a deep site (2-3 km) from which to drill/core through 121 C isotherm. Access to ancient groundwater (> 1 Ma, preferably >100 Ma)

27. More technical needs, desires, etc. Flowing water samples and/or core from deep sites (>1 kmbls) with mineralogy that may be conducive to abiotic, geochemical generation of H 2 (e.g., basalt, serpentinized ultramafic rock, Fe(II)-rich minerals, U- rich minerals). Flowing water samples and/or core from deep sites (>1 kmbls.) with evidence of biological sulfate reduction (significant H 2 S in ground water) or methanogenesis (significant CH 4 in groundwater or measurable partial pressure of CH 4 in localized areas of mine atmosphere). Biofilms from tunnel walls.

28. Technical requirements for geomicrobiological sampling: tracers –Solute: Br -, fluorochromes (e.g., rhodmine), perfluorinated hydrocarbons –Particulate: fluorescent carboxylated 1-µm microbeads core diameters >2 inches preferred drilling methods are highly site specific. anaerobic glove bag core barrels should be steam cleaned, core barrel liners freezer

29. DUSEL Geomicrobiology: Opportunities for new technologies Down-hole instruments Improved sampling and analyses of the geomicrobiology of rock-water interfaces Increased sensitivities for metabolic assays More sensitive geophysical approaches Metagenomics, proteomics, metabolomics

30. Applications of low-level counting in geomicrobiology: Dating groundwater, minerals, etc. Radiotracer experiments –Detecting and quantifying low rates of microbial activities –Imaging microbial processes –(However: limitation is likely the purity of commercially available radiochemicals, not counting sensitivities).

31. Radiorespirometry Low-level counting might enable lower-level abiotic controls, and thus greater sensitivity, but: –need higher purity radiochemicals –Non-biological oxidation of 14 C organic substrates can occur -- Mars Viking exp’t

32. Fluorescent in situ hybridization (FISH) and microautoradiography (MAR) or phosphorimaging Identify individual microbial cells by FISH –Sequence- specific genetic probe Test for substrate utilization in the same cells by MAR or phoshorimaging –Uptake of 14 C substrate