1. 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

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. State of Knowledge Low Diversity from a 2.825 km deep fault (Lin et al.,) 0.0 1.0 2.0 3.0 4.0 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Cells/ml or Cells/g Depth (km) 10 Pfiffner et al. 2006

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

6. Requirements of Subsurface Sampling Molecular 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 Sampling Sample Size 10 11 cells. (0.25 mg of DNA) ROCK –10 3 cells/g therefore need 10 8 grams!! WATER –10 3 cells/ml therefore need 10 5 liters (10,000L) At 1 gpm≈2 days If you have flowing water you can get good samples!

8. Requirements of Subsurface Sampling Location 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

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

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

13. How can we use Carbon Isotopes to Understand Subsurface Growth Rates? 14 C is generated in situ through decay of U and Th. 14 C in DIC, Hydrocarbons, CH 4 ….. 14 C in Microbes (DNA) Steady-state Production=Decay. Steady-state Production=Decay. No Production Only Decay after incorporation!

14. Imagining an Experiment Collect 14 C and 13 C of DNA, DIC, DOC and compound specific electron donors 14 C of DNA should be “older” with a more negative Δ 14 C The Δ 14 C 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 14 C microarray in a subsurface Beta Cage to relate specific genes to Δ 14 C

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

16. 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