PPROBINGROBING THETHE EEARTHARTH ’’SS DDEEPEEP BBIOSPHEREIOSPHERE !! T. Kieft, D. Moser, D. Boutt, H. Wang, L. Murdoch, C. Anderson, L. Stetler, S. Bang, R. Sani, B. Roggenthen, B. Mailloux, K. Nordstrom, B. Sherwood Lollar, J. Lippmann-Pipke, R. Stepanauskas, D. Emerson, S. Pffifner, T. Phelps, P. Long, K. Pedersen, T. Tokunaga, G. Slater, M. Conrad, H. Dong, E. vanHeerden, K. Takai and many others Communicated by T.C. Onstott 1010 1818 gg ofof livingliving CC onon EarthEarth • These microbes control biogeochemicalOcean cycling and fluxes between the surface and subsurface.Biosphere

• Both are crucial to climate change, CO 2 sequestration, long term waste storage and resource extraction. • The subsurface biosphere is relevant to the origin and evolution of terrestrial life and offers insights to the potential for life on other planets in our solar system. • The subsurface harbors diverse, novel and demonstrably useful, potentially ancient enzymes. • Current knowledge of the continental deep subsurface is limited, based on a few cores, fewer boreholes from the surface, still fewer deep mines and one URL.

Whitman et al. 1998 But none from the surface to the bottom of the biosphere

• “Subsurface Biosphere ” is a complex ecosystem requiring geological, geophysical, hydrological, geomechanical biogeochemical, molecular and microbiological characterization. • Over-arching question : What are 80 oC? the distributions and activities of 120 oC? subsurface life and what controls them? DeepDeep Biosphere:Biosphere: MajorMajor ResearchResearch QuestionsQuestions (from NSF S1 Deep Science)

1. How deeply does life extend into the Earth? 2. What fuels the deep biosphere? 3. How does the interplay between biology and geology shape the subsurface? 4. What are subsurface genomes telling us? 5. Did life on the earth's surface come from underground? 6. Is there life (down there) as we don't know it? Yet another 16S rRNA sequence – another butterfly Courtesy of G. Southam, UWO

2 µµµm 0.1 µm

• 16S rRNA gene libraries revealed this new species of bacteria was present only at depths >1.5 km and distributed over a distance of 300 km in the Witwatersrand Basin of South Africa • In some fractures it is the dominant species or only species. • You cannot grow it, but the16S rRNA gene sequence is very similar to sequences recovered from a deep sea vent • This species was remotely related to gram-positive sulfate reducing isolates so it might be a sulfate reducer. 2- Transport CrO 4 2- RSO O- 2+ MoO 4 2 Mg 2- CO 2- 2+ WO 4 3 Zn anions cations + 2+ + 2- 3- 2- - 3+ 2+ 2+ 2+ K Fe NH CrO lipo prot. export antimicrobial peptide efflux NO 4 4 polysacc. export multidrug export PO SO 3 Co Mn

Fe Ni polar AA 4 4 dipeptide and other AA branched chain AA type IV sugars pilus

gas - ATP ADP ATP 2H + CO +H O O2 + 2 2 H+ H superoxide dismustase + P i NH vesicle Nitrogenase 3 Na + H2O2 N2 AA, NA, NAD organic 2Na + rubrerythrin synthesis synthesis 2+ H2O Ca NH 3+ H 2 gln 8e -+ ADP Gln synthetase HCOO - Pentose phosphate HPK(9) Flagella 18ATP + P pathway i +THF RR(19) Formate DH Glycolysis Signal +THF-CH 3 +CoA Gluconeogenesis MCP(5) Sec-dep + Acetyl-CoA 2- 2- 2- - CO protein SO 4 APS-SO 4 SO 3 HS 2 CO Acetyl-CoA endospore synthase Transduction Sec-indep ADP protein Citric acid + P LDH - - HCOO - ATP i e e CO 2+H 2 2H + 2e - 2H + 2e - Cycle CO 2 2H + 2e - F420 Fe, Ni Fe FoF1-ATPase CO 2 Lactate Qmo Hme SDH

+ + Formate 3- + 2- H H H H H H+ Na +? PO 4 Na SO 4 hydrogenlyase 2 2 2 complex ATP 2.3 Mb with 2241 ORF's Synthesis Chivian et al. 2007

WhatWhat doesdoes thethe genomegenome telltell us?us? WhatCandidate does speciesthis subsurface - Desulforudis genome audaxviator tell us? Courtesy of G. Southam, UWO

2 µµµm 0.1 µm

"In Sneffels Joculis craterem quem delibat Umbra Scartaris Julii intra • No protection against O 2 but does against H 2O2 • Bothcalendas heterotrophic descende, and autotrophic, Audax adaptable viator to, aet changing terrestre environment centrum attinges.” • ("Descend,Chemotactic, mobilebold travellerthrough fractures, into where the crater environ mentof the may Jokul vary of Sneffels, which • Can fix N 2 if need be, when energy resources are high •theCan shadow sporulate ofif need Scartaris be, when touches energy resources before are the low kalends of July, and you will • Possesses all the machineryattain for a solitarythe center life, se oflf-reliant the earth.”) • Very-- Hidden low SNP’s, message almost clonal, deciphered suggesting fromlow mutat an ionIcelandic rates saga that prompts • Contains genetic elements transferred from anaerobic, thermophil ic methanogens (i.e. HGT), did Professorthis happen Lidenbrockin the subsurface? to undertake his travels in Jules Verne’s “Journey • Contains CRISPR sequences,to protection the Center again ofviral the infections? Earth” 2- CrO 4 2- RSO O- 2+ MoO 4 2 Mg 2- CO 2- 2+ WO 4 3 Zn anions cations + 2+ + 2- 3- 2- - 3+ 2+ 2+ 2+ K Fe NH CrO lipo prot. export antimicrobial peptide efflux NO 4 4 polysacc. export multidrug export PO SO 3 Co Mn

Fe Ni polar AA 4 4 dipeptide and other AA branched chain AA type IV sugars pilus

gas - ADP ATP + O2 ATP + 2H CO 2+H 2O H + superoxide dismustase + P i NH H vesicle Nitrogenase 3 Na + H2O2 N2 AA, NA, NAD organic 2Na + rubrerythrin synthesis synthesis 2+ H2O Ca NH 3+ H 2 gln 8e -+ ADP Gln synthetase HCOO - Pentose phosphate HPK(9) Flagella 18ATP + P pathway i +THF RR(19) Formate DH Glycolysis Signal +THF-CH 3 +CoA Gluconeogenesis MCP(5) Sec-dep + Acetyl-CoA 2- 2- 2- - SO APS-SO SO HS H SH+ CO 2 protein 4 4 3 2 CO Acetyl-CoA Transduction endospore sythase Sec-indep ADP protein Citric acid + P LDH - - HCOO - ATP i e e CO 2+H 2 2H + 2e - 2H + 2e - Cycle CO 2 2H + 2e - F420 Fe, Ni Fe FoF1-ATPase CO 2 Lactate Qmo Hme SDH

+ Formate 3- + H+ H + +? PO Na SO 2- hydrogenlyase H H H H Na 4 4 CO 2 2 2 2 complex + H2O Na ) + 2- H O H2 OH 3 4H 2SO 4 2 2 Fe( H2 2H + 2H 2O - HCO2+ 3 - Ca HCO 3 2+ 2H 2O Ca

2H O + 2 2H ) 2 H2O2 H α, β, γ (O 0 FeS 1 2 O i 4 CaCO 3 S NaAlSi 3O8 U2O4 g 3 M 2- Transport CrO 4 2- RSO O- 2+ MoO 4 2 Mg 2- CO 2- 2+ WO 4 3 Zn anions cations + 2+ + 2- 3- 2- - 3+ 2+ 2+ 2+ K Fe NH CrO lipo prot. export antimicrobial peptide efflux NO 4 4 polysacc. export multidrug export PO SO 3 Co Mn

Fe Ni polar AA 4 4 dipeptide and other AA branched chain AA type IV sugars pilus

gas - ADP ATP + O2 ATP 2H CO 2+H 2O superoxide dismustase + P i NH vesicle Nitrogenase 3 Na + H2O2 N2 AA, NA, NAD organic 2Na + rubrerythrin synthesis synthesis 2+ H2O Ca NH 3+ H 2 gln 8e -+ ADP Gln synthetase HCOO - Pentose phosphate HPK(9) Flagella 18ATP + P pathway i +THF RR(19) Formate DH Glycolysis Signal +THF-CH 3 +CoA Gluconeogenesis MCP(5) Sec-dep + Acetyl-CoA CO 2 protein H2S CO Acetyl-CoA Transduction endospore sythase Sec-indep ADP protein Citric acid + P LDH - - HCOO - ATP i e e CO 2+H 2 2H + 2e - 2H + 2e - Cycle CO 2 2H + 2e - F420 Fe, Ni Fe FoF1-ATPase CO 2 Lactate Qmo Hme SDH Formate 3- PO 4 hydrogenlyase H2 H2 H2 2- complex SO 4 H) 3 + e(O - H F HCO 2 - HCO2+ 3 - Ca HCO 3 2+ 2H 2O Ca

H+ ) 2 H (O 0 FeS 1 2 O i 4 CaCO 3 S NaAlSi 3O8 U2O4 g 3 M Large Volumes of Water Help – Particular when searching for rare, but important organisms

ButBut doesdoes thisthis telltell usus howhow biologybiology shapesshapes thethe subsurfacesubsurface oror whatwhat fuelsfuels thethe deepdeep biosphere?biosphere? DeepDeep BiosphereBiosphere Research:Research: CurrentTheCurrentThe NextNext StateState GenerationGeneration ofof AffairsAffairs

10 8

Rockc 10 6 ores Cells/gram Cells/gram pmols/gm-yr

Water Single Cell 10 4 Genomics

Who is there. 10 2 RNA WhatProtein they are doing. Trace organic species 1 10 RNA 100 1000 Depth (m) 5000 TransformativeTransformative sciencescience goals,goals, whywhy isis DUSELDUSEL required?required?

• What are the microbes doing? Perform experiments in Thisa geologically requires a andcoordinated, geochemically multi-institutional, well-characterized multi- disciplinary,environment. experimental facility at at least one location focused on the biosphere, from the surface to the base, for • Why are they doing what they are doing? Perform in multiple decades. It is the in situ experimentation that will situ experiments under a variety of environmental develop a deeper understanding of the complex and stimulus (including changes in temperature, stress and interrelated phenomena surrounding subsurface life. flow rates). • How did they come to be there? Characterize the ecohydrology from the surface to the base of the biosphere. In situ P&T borehole experiments • Monitor changes during induced near- field seismic events • Circulate various labeled metabolites into the borehole and monitor uptake kinetics, growth rates, etc.

176x30 Autosampler MULMUL ’’ss –– MobileMobile UndergroundUnderground LaboratoriesLaboratories

Äspo Hard Rock Laboratory Why Homestake? – Geologically well-characterized 3D volume to deepest levels in USA.

Should provide access to a variety of ground water ages from the top to the bottom of the subsurface biosphere.

AreHow doCritically Microbial Stressed Communities Faults Respond Critical to Hydrotherma to Subsurfal Circulation?ce Life?

• AreDuring thermophiles fault rupture enriched do changes as mesophiles in fluid flux die? induc e • Doeschanges enhanced in chemical fluid fluxflux whichlead to in enhanced turn alter activity andmicrobial growth dynamics? rates? • If so, then is this an important process for the b La es ess subsurface biosphere? oc Pr led The fault with no name. up Connecting Co Drift Observation Drift

Heated Drift Wing Heaters Thermal Mechanical Hydrological Chemical Looking Down on the LM150 Drill 7400’ Deep Microbiology and Pad Facility – three boreholes down to 5+ km – 120 oC

Drill Rod Cleaning Station

Tracer injection Water filtration system

Cuttings Tank Generators MUL >100 Size 75 PORE THROAT SIZE HISTOGRAM Classifications: 25 macro 7.5

2.5

0.75 meso

0.25 micro 0.075

Pore Throat Radius, microns Pore Throat 0.025

0.0075

<0.0025 0.0 A. Mercury Saturation, fraction (frequency) D.

B. C. Low Background Counting Laboratory Located at the 4850’ Level

Lab space for preparation of α, β and γ biological and counters behind hydrological water shield, samples (e.g. including large 210 Pb, 137 Cs, volume β cage. 39 Ar, etc.)

Ecohydrology would produce ~60,000 samples for counting over 5 years Conceptual design of the DUSEL biology program 1. MREFC - Clean coring and core hole completions from surface to base of biosphere with metal-free packers isolating individual fractures. a. Employ proven and new technologies to characterize geochemistry and microbiology of rock matrix, fracture surfaces and fracture water. b. Monitor fractures over time until they stabilize. 2. Post MREFC - Once core hole has recovered from drilling impact initiate experimental phase. a. Perform single cell DNA sequencing to generate reference genomes for community DNA, RNA and protein studies b. Perform population genomics analyses to determine evolutionary histories, current selective pressures and gene flow. c. Utilize MUL’s to perform microcosm and in situ experiments that employ isotope labels and isotope microarrays to quantify metabolite flow, respiration rates, replication rates, the effects of nutrient limitation, quorum sensing, viral populations, and predation. IntegrationIntegration ofof biologybiology programprogram withwith otherother activitiesactivities atat DUSELDUSEL FAARM

LUCI – Geophysics CO 2

EcohydrologyEcohydrology

Induced THMCB Fracture Lab Sanford Center NetworkLocations of Inner of Deep Space Continental Observatories Subsurface (NISO) proposed by MicrobialBarbara Sherwood Investigations Lollar, Univ. Toronto

Phyäsalmi URL ASPO HRL Olkiluoto Canadian Au Songliao mines Mt. TERRI URL DUSEL SNOLab

LSBB URL Mizunami URL WIPP

NELSAM

> 100 m > 1km Oil field well head studies New sites being developed LUCI ASPO

THMBC

IF AnticipatedAnticipated BiologicalBiological ResultsResults fromfrom DUSELDUSEL inin thethe ComingComing DecadeDecade

• Discovery of the ultimate high temperature limit for life in the crust and what controls it.

•Discovery of what controls the “average age” of microorganisms in the deep subsurface.

•Discovery of what other factors control subsurface biomass concentration, diversity, migration rates and evolution.

•Discovery of how microorganisms impact rock strength, hydrothermal circulation and CO 2 storage by mineral dissolution and precipitation.

•Discovery of new geophysical sources of energy for deep subsurface microbial communities.