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Brian J. Mailloux Barnard College For the Sloan Deep Carbon Workshop May 16, 2008

Subsurface Microbial Carbon Cycling: Rates and Processes or Recovery and Characterization of a Deep Microbial Ecosystem. Brian J. Mailloux Barnard College For the Sloan Deep Carbon Workshop May 16, 2008. Talk overview. Background Sampling Requirements Use of Carbon isotopes.

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Brian J. Mailloux Barnard College For the Sloan Deep Carbon Workshop May 16, 2008

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  1. Subsurface Microbial Carbon Cycling: Rates and ProcessesorRecovery and Characterization of a Deep Microbial Ecosystem Brian J. Mailloux Barnard College For the Sloan Deep Carbon Workshop May 16, 2008

  2. Talk overview • Background • Sampling Requirements • Use of Carbon isotopes

  3. State of Knowledge • Examining depths to 120°C • Lower cell numbers at greater depth • Lower diversity at greater depths • Slow • Hard to sample Can we use carbon isotopes to understand rates and turnover times and in the future link to diversity?

  4. Cells/ml or Cells/g 10 102 103 104 105 106 107 108 0.0 1.0 Depth (km) 2.0 3.0 Pfiffner et al. 2006 4.0 State of Knowledge Low Diversity from a 2.825 km deep fault (Lin et al.,)

  5. Requirements of Subsurface SamplingConstraints • CLEAN • Molecular sample constraints? • Sample Size-How large a sample do we need? • Location-Where and how can we sample?

  6. Requirements of Subsurface SamplingMolecular Constraints • PCR • Nanograms of DNA • Metagenomes • 10’s to 100’s of micrograms of DNA • Amounts can be lower with whole genome amplification • Isotopes • 100’s of micrograms of DNA • PLFA’s generally have smaller sample sizes than DNA Knowledge DNA

  7. Requirements of Subsurface SamplingSample Size • 1011 cells. (0.25 mg of DNA) • ROCK • 103 cells/g therefore need 108 grams!! • WATER • 103 cells/ml therefore need 105 liters (10,000L) • At 1 gpm≈2 days • If you have flowing water you can get good samples!

  8. Requirements of Subsurface SamplingLocation • Cores • Access to novel locations • Expensive and size limited • Wells • Access to novel locations • Deep wells can be hard to sample • Mines • Access to the subsurface • Locations limited • Can get clean samples • Can go back repeatedly and run experiments

  9. Carbon Isotopes of DNA • Bangladesh Example • How it could be used in the deep subsurface • 12C=99%, 13C=1%, 14C=1ppt but t1/2=5730 yr • Microarrays

  10. Analyzing 14C of DNA Bangladesh ExampleAtmospheric derived 14C • Sampled ~2000 liters from a 180’ deep well. • Extracted DNA ~150μg (Not trivial!) • 14C DOC ~5700 yr bp • 14C DIC ~6240 yr bp • 14C DNA ~300 yr bp Small, Young, Labile Pool of Organic Carbon! E. Reichert, Senior Thesis

  11. Steady-state Production=Decay. Steady-state Production=Decay. No Production Only Decay after incorporation! How can we use Carbon Isotopes to Understand Subsurface Growth Rates? 14C is generated in situ through decay of U and Th. 14C in DIC, Hydrocarbons, CH4….. 14C in Microbes (DNA)

  12. Imagining an Experiment • Collect 14C and 13C of DNA, DIC, DOC and compound specific electron donors • 14C of DNA should be “older” with a more negative Δ14C • The Δ14C offset should be directly related to the turnover rate (“age”) of the microbes. • Can then directly get to turnover times in the deep subsurface. • Can then use a 14C microarray in a subsurface Beta Cage to relate specific genes to Δ14C

  13. Goals-Need to Link • Isotopes • Geochemistry • Genomics/Proteomics • With good subsurface access

  14. Conclusions ACKNOWLEDGEMENTS T.C. Onstott and collaborators within his lab including: Dylan Chivian, Eric J. Alm, Eoin L. Brodie, David E. Culley, Thomas Gihring, Alla Lapidus, Li-Hung Lin, Steve Lowry, Duane P. Moser, Paul Richardson, Gordon Southam, Greg Wanger,Lisa M. Pratt, Adam P. Arkin, Terry C. Hazen, Fred J. Brockman, Duane Moser Columbia University- Greg Freyer, Martin Stute, Lex van Geen, Elizabeth Reichert LLNL-Bruce Buccholz

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