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13 C and 14 C Studies of Microbial Carbon Cycling in the Deep Subsurface

+. -. 13 C and 14 C Studies of Microbial Carbon Cycling in the Deep Subsurface. Kevin Mandernack Dept. of Chemistry and Geochemistry Colorado School of Mines Golden CO 80401 kmandern@mines.edu. Importance of Life in the Subsurface. Groundwater Resource 1

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13 C and 14 C Studies of Microbial Carbon Cycling in the Deep Subsurface

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  1. + - 13C and 14C Studies of Microbial Carbon Cycling in the Deep Subsurface Kevin Mandernack Dept. of Chemistry and Geochemistry Colorado School of Mines Golden CO 80401 kmandern@mines.edu

  2. Importance of Life in the Subsurface • Groundwater Resource1 • 30% of freshwater is groundwater (0.3% lakes and rivers) • Deep pristine groundwater is threatened by percolating contaminants • Radioactive Waste Disposal2 • 131 temporary sites / 39 states • 105,000 metric tons spent nuclear reactor fuel in U.S. by 2035 • Origins of Life / Extraterrestrial Life • Could life have originated underground?3,4 • Could there be life in the deep subsurface of Mars?3,5 1World Meteorological Organization (http://www.wmo.ch) 4Pedersen (1997) FEMS Microbiology Reviews 2Department of Energy (http://www.ocrwm.doe.gov) 5McKay et al. (Science) 1996 3Stevens (1997) FEMS Microbiology Reviews

  3. Research Overview • Microbes control subsurface aqueous geochemistry Statement: Objective: • Characterize microbial communities & carbon cycling in the subsurface Approach: • Structural analysis of bacterial cell membrane phospholipid-derived fatty acids (PLFAs) • Measure d13C and 14C values of bacterial PLFAs • Measure the d13C and 14C values of possible groundwater carbon sources (DOC, DIC, POM, CH4)

  4. Bacterial Metabolisms: Heterotrophy Autotrophy Methanotrophy

  5. Subsurface Bacterial Metabolisms

  6. Summary of Terminal Electron Accepting Processes Sulfate Reduction Fe(III) Reduction Methane Production Aerobic Respiration Mn(IV) Reduction Denitrification Organic Matter Oxidation CO2 C2H4O2 C2H4O2 C2H4O2 CO2 C2H4O2 CO2 C2H4O2 CO2 CO2 CO2 C2H4O2 SO42- 2O2 1.6NO3- 8FeOOH 4MnO2 4MnCO3 8FeCO3 1.8N2 2H2O CH4 S2- Free Energy (kJ / mol e-) -6 -77 -22 -4 -106 -98 SO42- Fe(III) Mn(IV), Mn(II) NH4+, N2 S2- Fe(II) Mn(II) NH4+, N2 SO42- Fe(III) Mn(IV) NO3-, N2 SO42- Fe(III), Fe(II) Mn(II) NH4+, N2 SO42- Fe(III) Mn(IV) NO3-, N2 SO42-, S2- Fe(II) Mn(II) NH4+, N2 Predominant Redox Species 10 Characteristic Hydrogen Concentration Not Applicable (nM) 5 (<0.1 nM) (<0.1 nM) 0 Lovley and Chapelle (1995) Reviews of Geophysics

  7. Typical Distribution of Terminal Electron Accepting Processes in Deep Aquifers Lovley and Chapelle (1995) Reviews of Geophysics

  8. Two Hypotheses for Abiotic H2 Production in the Subsurface Radiolysis of Water by Uranium Decay1 235U 231Th a2+ H2 + ½O2 H2O Water-Basalt Interaction2 (X/2)H2O + (FeO)X(SiO2)y (X/2)H2 + X(FeO3/2) + Y(SiO2) basalt (mafic) 1Pedersen (1997) FEMS Microbiology Reviews 2Stevens & McKinley (1995) Science

  9. Photosynthesis-Independent Subsurface Ecosystem H2 Oxidizers Desulfobacterium(H2 + CO2) Desulfovibrio (H2 + acetate) Shewanella (H2 + org. C source) Organic Polymers Acetate Oxidizers Geobacter Desulfuromonas Desulfobacter Methanotrophs Fermenters Propionibacterium Clostridium CH4 CH4 acetate acetate autotrophic acetogenic bacteria acetoclastic methanogens autotrophic methanogens H2 CO2 “geogas” Pedersen (1997) FEMS Microbiology Reviews

  10. Identification of Bacteria by Phospholipid Fatty Acid (PLFA) Analyses

  11. + - Archaea (phosphoether lipids) + - Bacteria cell membrane (phosphoester lipids) CH3 CH3 CH3 CH3 CH3 Taylor & Parkes (1983) Journal of General Microbiology Bowman et al. (1991) FEMS Microbiology Ecology Kohring et al. (1994) FEMS Microbiology Letters Pinkart et al. (2002) In: Manual of Environmental Microbiology Guckert et al. (1991) Journal of General Microbiology

  12. Schematic of Site Geology & Sampling SE ~2 km Tono Mine Shaft Seto Group MSB-2 Akeyo Form. Odiwara Form. Tono Uranium Mine Upper 3 Toki, JAPAN Toki Lignite- Bearing Form. Lower Lignite Seams 4 Mine Gallery Uranium Ore Bodies Iwatsuki et al. (2001) Applied Geochemistry 1 5 Iwatsuki & Yohsida (1999) Geochemical Journal 2 KNA-6 Tsukiyoshi Fault Artesian Flow Sasao et al. (2006) Geochemistry: Exploration, Environment, Analysis Toki Granite

  13. Experimental Procedures 3. 2. Total Lipids 1. Cells on Filters Phosphoplipid Isolation Via SPE Bligh-Dyer Total Lipid Extraction Filtration (0.2 mm) of Groundwater or Bacterial Culture Phospholipids 5. Structural Information a) GC-MS 4. MeOH + KOH + Heat b) GC-Isotope Ratio MS d13C Fatty Acid Methyl Esters (FAMEs) c) Accelerator MS 14C Values Mild Alkaline Methanolysis Mass Spectrometer Analyses

  14. PLFA Profiles of Tono Area Groundwaters Type II methanotroph MSB-2 Granite i14:0 14:0 i15:0 ai15:0 15:0 i16:0 16:0 16:1w7c 16:1w7t 16:1w5c cy17:0 18:0 18:1w9c 18:1w8c 18:1w7c 18:2 (n6) KNA-6 Sedimentary % of Total PLFA KNA-6 Granite PLFA Structure

  15. PLFA Profiles of Henderson Mine Groundwaters Sulfate reducers? D-4 (higher SO42-) trace 14:0 me15:0 i15:0 ai15:0 15:0 br16:0 16:0 16:1w7 10me16:0 i17:0 ai17:0 cy17:0 18:0 18:1w7 18:1w9 18:1w11 D-1 % of Total PLFA trace D-3 3/9/06 Pre-packer PLFA Structure

  16. Comparison of PLFA-Based and Direct Cell Counts MSB-2 Granite KNA-6 Sedimentary KNA-6 Granite PLFA-Based Total Cells D-4 3/23/06 Direct Count Total Cells D-3 3/9/06 PLFA-Based Methanotrophic Cells D-1 3/23/06 Log [cells per mL]

  17. Determination of Bacterial Diet by d13C and 14C of PLFAs

  18. 12CH2O 12CO2 12CO2 12CO2 12CH2O 12CO2 12CO2 12CH2O 12CH2O 12CO2 12CO2 12CH2O 12CO2 12CH2O 12CO2 12CH2O 12CH2O 12CH2O 12CH2O 12CH2O 12CH2O 12CO2 13CO2 13CO2 13CO2 13CO2 13CH2O 13CH2O Stable Carbon Isotope Fractionation Many biologically-mediated reactions prefer 12C to 13C due to slightly smaller bond strengths d13CCO2 = -9 ‰ CO2 Fixation d13Cbiomass = -27 ‰ D13Cbiomass-CO2 = d13Cbiomass - d13CCO2 = -18‰

  19. Stable Carbon Isotope Signatures of Carbon Assimilation Pathways More Depleted d13C Values of Biomass Heterotrophy Autotrophy Methanotrophy Organic Compounds (CH2O) Energy CO2 CH4 + CO2 Energy + CO2 + Calvin Benson Others Type II & X Type I, II, & X Energy D13C = ~0 RuMP D13C D13C Serine = = D13C D13C Biomass Carbon -10 to -22 0 to -36 = = -14 to -29 -5 to -14 Biomass Carbon Biomass Carbon PreuB et al. (1989) Zeitschrift für Naturforschung Summons et al. (1994) Geochimica et Cosmochimica Acta Jahnke et al. (1999) Geochimica et Cosmochimica Acta Hayes (2001) in: Stable Isotope Geochemistry, Reviews in Mineralogy and Geochemistry

  20. d13C and 14C – Two Indicators of Carbon Source Atmospheric CO2 100 Modern Organic Matter Modern Carbonates 80 60 14C (pMC) 40 20 Ancient Organic Matter Ancient carbonates 0.1 +10 -20 -10 0 -30 marine carbonates1 atm CO21 C3 plants2 Algae2 C4 plants2 -110 Methane4 freshwater carbonates1 d13C (‰) 1Hoefs (1997) Stable Isotope Geochemistry 3Feux (1977) Journal of Geochemical Exploration 2Faure (1986) Principles of Isotope Geology 4Whiticar et al. (1986) Geochimica et Cosmochimica Acta

  21. Isotopic Values of Carbon Sources in KNA-6 Groundwaters Groundwater Transport d13C (‰) 14C (pMC) Carbon Source

  22. 14C and d13C Values of PLFAs From Tono Area Groundwaters PM14C = 57 Drill Fluid PLFA MSB-2 Borehole 16:0 79-130 m SEDIMENTARY 16:1 suite MSB-2 Borehole PM14C = 59 cy17:0 132-154 m 18:1 suite PM14Ctotal PLFAs = 33 Tono Mine MSB-2 Borehole 171-175 m GRANITE Tono Mine -60 -50 -40 -30 -20 -10 0 13C (‰ PDB)

  23. Conclusions 1. PLFA analysis indicates diverse bacterial communities in granite-hosted ground waters at Tono & Henderson Mines. Results at Henderson suggest the presence of sulfate reducing Bacteria. 2. Estimates of cell numbers at Henderson are ~1x104/ml. • d13C values of bacterial PLFAs are generally lower for the granite rocks relative to sedimentary rocks at Tono Mine, suggesting that autotrophic metabolism, including methanotrophy, is more prevalent here. Henderson???

  24. Acknowledgements • Chris Mills & Raleigh Schmidt – CSM • Teruki Iwatsuki, Ph.D. – JNC Tono Geoscience Center, Japan • Yuki Murakami, Ph.D. – JNC Tono Geoscience Center, Japan • Bob Dias, Ph.D. – Dept. of Chemistry, Old Dominion University, Norfolk, VA • Greg Slater, Ph.D. – School of Geography & Geology, McMaster University, Hamilton, ON • Chris Reddy, Ph.D. – Woods Hole Oceanographic Institution, Woods Hole, MA Funding • Japan Nuclear Cycle • Earth Sciences program of NSF (EAR-9985234)

  25. Remaining Questions • Do aerobic methanotrophs and methanogens coexist? • Where do aerobic methanotrophs get O2 in a reduced environment? ? ? ? ? ? ? ? ? ?

  26. Conclusions 1. d13C values of bacterial PLFAs are generally lower for the granite rocks relative to sedimentary rocks, suggesting that autotrophic metabolism, including methanotrophy, is more prevalent here. 2. Detection of the diagnostic 18:1w8 PLFA and molecular evidence for a monooxygenase enzyme indicate that type II methanotrophs are present in Tono mine groundwaters. 3. d13C value of CH4 suggests autotrophic methanogenesis in KNA-6 groundwaters 4. Initial 14C analyses of bulk bacterial PLFAs from Tono Mine water indicate bacteria indeed utilize relatively old carbon (~8,900 YBP)

  27. Additional d13C and 14C Values of Subsurface Carbon

  28. Examples of Archaebacterial & Bacterial Lipids Sulfate-Reducers Archaebacteria Lipids Desulfovibrio sp. crocetane (2,6,11,15-tetramethylhexadecane) iso-C15:0 fatty acid anteiso-C15:0 fatty acid PMI (2,6,10,15,19-pentamethylicosane) iso-C17:1w7 fatty acid Methanotrophs Desulfobacter sp. C16:1w8 (type I) 10-methyl C16:0 cyclic C17:0 C18:1w8 (type II) Makula, 1978; Coleman et al., 1993; Vainshtein et al., 1992; Nicholas et al., 1986; Chappe et al., 1982

  29. Separate individual compounds on capillary GC column Collect selected compounds in cryogenic traps connected to column by a computer controlled zero-dead-volume multiport valve Repeat injection up to 100 times to collect sufficient sample for 14C analysis ( 100 mg C) Autoinjector required for retention time consistency Isolation of Specific Compounds for 14C Analysis (preparative capillary gas chromatography) Eglinton and Aluwihare, Analytical Chemistry, 1996.

  30. D14C of PLFAs Indicates Kerogen as Carbon Source PLFA Values of Kerogen Enrichment Culture Petsch et al., 2001

  31. 13C/12C = 1.11 % PeeDee Belemnite (PDB): 13C/12C = 0.0112372 atom % 13C = 13C / (12C + 13C) d13C = (Rsam - Rstd) x 1000 Rstd 13C = 1,001 13C = 1,000 12C = 100,000 d = +1 ‰ 12C = 100,000 Rsam = 0.01001 Rstd = 0.01000

  32. Phospholipid Fatty Acids (PLFAs) of the Cell Membrane aqueous cell exterior hydrophobic membrane interior + - aqueous cell interior Cell Death (phospholipase) OH J.D. Hendrix http://science.kennesaw.edu White & Ringleberg, 1997

  33. Dating Subsurface Carbon Sources With 14C Atmospheric 14C ~ 1ppt of total C 1/2 life = 5730 years Percent Modern Carbon (pmc) Age (Kyears Before Present) www.NOSAMS.WHOI.edu Faure, 1986

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