1. Fine scale control of microbial communities in deep marine sediments that contain hydrates and high concentrations of methane F.S. Colwell, B. Briggs, P. Carini, M. Torres (Oregon State Univ) M.E. Delwiche (Idaho National Laboratory) E. Brodie (Lawrence Berkeley National Laboratory) R. Daly (UC Berkeley) A. Hangsterfer, M. Kastner (Scripps) M. Holland (Geotek Ltd.) P. Long, H.T. Schaef (Pacific Northwest National Laboratory) W. Winters (USGS Woods Hole) M. Riedel (McGill Univ) Acknowledgements: U.S. Department of Energy, Office of Fossil Energy, National Energy Technology Lab; Integrated Ocean Drilling Program, Science Parties from Leg 204 & NGHP Offshore India

2. How does fine-scale variability in marine sediments control the number, type and distribution of microbial communities (methanogens, heterotrophs)? --> Refine computational models with biological rate terms that are consistent with sediment conditions. Approach: Microbiology Methanogen enumeration (OSU); molecular ecology by PhyloChip, t-RFLP, and clone library (LBNL/UCB, OSU, Scripps) Geology/chemistry Temperature, IR imagery (PNNL); grain size analysis (USGS WH); porewater chemistry (OSU; Scripps); hydrocarbons, C-isotope ratios, hydrogen (USGS) Multivariate analysis Nonmetric multidimensional scaling Hydrate No hydrate M = Microbiology G/S = geochem, sedimentology, etc. 0 mbsf 100 mbsf geochemistry, sedimentology microbiology geochemistry, sedimentology microbiology

3. 17A 19A 46 microbiology cores 31 hydrate; 15 non-hydrate Mostly KG Basin (passive margin) 12 from Andaman Islands (deep hydrates, convergent margin) 15A Cl- temperature IR images

4. DNA extractions (Scripps, OSU) PCR on DNA extracted from India samples Diluted DNA is more likely to amplify: –correct concentration? –dilution of inhibitors? Bacterial vs. archaeal DNA is: –more prevalent? –more easily extracted? –more easily amplified?

5. Terminal restriction fragment length polymorphism (T-RFLP) from Gruntzig et al. 2002 PhyloChip -500,000 probes 300,000 target 16S genes 2 domains --> 9000 taxa

6. t-RFLP relative fluorescence 21A78.122 14A10316 20A115.312 20A120.721 Site Depth (mbsf) Phylotypes t-RFLP perfomed on DNA amplified using archaeal primers and HaeIII as the restriction enzyme No matches with t-RFs entered in the MSU RDPII database

7. Archaea - Key observations: -methanogens, ANME-1 present -High temperature archaea most abundant in 10D-4H -Low temperature methanogen most abundant in 10D-10X -non-hydrate samples are more similar to each other than to hydrate samples; however, not very similar -ANME-1 has highest intensity in 10D-4h (shallowest sample) 3B-20X 10D-4H 10D-10X 14A-19X 200-800 taxa detected

8. Bacteria - Key observations: -Sulfate reducers, sulfur oxidizers, ANAMMOX, acetogens, aerobic methanotrophs, metal reducers 3B-20X 10D-4H 10D-10X14A-19X

9. An unusual microbial community in methane- rich sediments? pink/orange slime fractures; 0.5-20 mbsf large coccoid cells, often as tetrads above the SMI Offshore India Hydrate Ridge Offshore Vancouver Island (Riedel et al. 2006) Ca. 30 um

10. Summary Fine-scale control of microbial communities: Preliminary molecular data suggests numerous taxa detected including: –Archaea: methanogens, anaerobic methanotrophs, thermophiles –Bacteria: sulfate reducers, sulfur oxidizers, metal reducers, ANAMMOX, acetogens, aerobic methanotrophs C, S, N, and metal biogeochemical cycles possible Next… Multivariate analysis of communities; reconcile with geochemistry, geology; determine controlling factors Determine identity and biogeochemistry of intriguing shallow biofilms Relationship of methanogenic rates to molecular biology data

11. Steady-state view of the global carbon cycle (Dickens, 2003) Accurate estimates to come from temporal modeling of CH 4 inputs and outputs in appropriate hydrate environments CH 4 inputs are poorly understood Realistic rates of methanogenesis? Methanogen locations and controlling factors? Other biogeochemical processes to sustain the system? 4H 2 + CO 2 CH 4 + 2H 2 O CH 3 COOH CH 4 + CO 2