1. Initial Geochemical and Microbiological Characterization of Henderson Fluids How does knowledge of the site-specific chemistry at Henderson enhance our ability to identify subsurface microbial organisms (phylogentically and functionally) -- and their direct dependency upon fluid-rock interaction? BIOSCIENCE SAMPLING TEAM Templeton: Henderson DUSEL Capstone Workshop May 4-7th Tom Kieft (New Mexico Tech) for Alexis Templeton (University of Colorado – Boulder)

2. Fluid Chemistry: 7025 level Warm, ~40C; pH 5.9 to 6.2 Dissolved O 2 : 0.3 to 3.8 mg/L High DIC (~30 mM) High Mn 2+, Fe 2+ (1 to 20 mM) High SO 4 2- (4 to 48 mM) Trace metals: Zn > Ni > Mo Low (but detectable) organics (50  M) Abundant nitrogen species: includes NO 3 -, NO 2 -, N 2 O, NH 4 + Flowing boreholes variably mixed with oxygen O 2 concentrations increase as borehole flow-rate decreases From D1  D3 O 2 levels drop 1000x within 1 hour of packer-insertion D1 D2 D3 Establishing the basic chemistry, nutrient levels and cell numbers in deeply-sourced, hot fluids:

3. Natural Fracture Surface: Degassing CO 2 Can also detect N 2 O (6-18 microM), only low CH 4 (sub-microM), and H 2 not measured yet. CO 2 Movie: High CO 2 important for autotrophic growth

4. Fe-oxides Sulfates Fe 2+ rapidly oxidizes and oxide and sulfate minerals precipitate Fe 2+ rapidly oxidizes and oxide and sulfate minerals precipitate New minerals hard to structurally identify – relatively amorphous What’s the disequilibrium between the fluids and the surface? Oxic-Anoxic Interface

5. Adjacent to the Fracture: Abundant Mn(IV)-oxides form black mineral coatings on the tunnel walls. LBB-test Shows Mn(IV) In minerals; Mn(II) in fluids

6. First assessement: Microbial Diversity in the Ancient Fluids Data from John Spear, CSM Surprising abundance of Eukaryotic fungal sequences..

7. Second assessment: Microbial Diversity in fluids of varying chemistry (samples only separated by meters….) More dilute fluids (7025-D1), pre-packer: Dominated by uncultured beta-Proteobacteria (e.g. Japanese Gold Mine): 80% Fluids with high Fe, Mn, sulfate, NH 4 +, N 2 O (7025-D4) post-packer: Abundant delta-Proteobacteria (SRB) Abundant Nitrospira Remarkable diversity (~27 other groups) See John Spear talk on Friday…

8. Thermophilic Metal-oxidizing bacteria? Microbes more abundant in the Fe-oxide mats Rapid growth on Mn-media at 50°C using D4 mineral mat! First 3 isolates being sequenced Water and Mat samples:  Fe-oxidizing bacteria  Mn-oxidizing bacteria Establish enrichments: *site-specific Henderson medium* with and without organics near-neutral pH stabilized on mineral surfaces culture at 40°C-55°C Growth on Fe-media also successful… DNA-labeled cells Fe-oxide particles

9. Dissolved Oxygen now below detection Now target anaerobic enrichments: Isolates for biochemical and proteomic studies Anaerobic Fe-oxidizers Mn-reducers Fe-reducers Sulfate-reducers In-situ biofilms recently extracted for analysis

10. Summary to-date Yes, microorganisms are present in the fluids, at low cell numbers. The fluid chemistry at Henderson is unique due to water-rock interaction, which has implications for the likely modes of metabolism and enzymatic activities. –CO 2 and sulfate concentrations are very high –N and C nutrient levels are above detection limits, but not P –Millimolar concentrations of Mn, Fe and Zn Fluid chemistry will vary significantly depending upon location inside vs. outside mineralized rock. The microbial diversity is high and includes unexpected fungal sequences There exists unexpected potential for a subsurface microbial N-cycle NH 4 +, NO 2 -, NO 3 - and N 2 O all abundant (naturally or not?) Microbial organisms are more abundant at the oxic-anoxic interface, where the Fe- oxide mineral mats are forming. Initial culturing efforts targeting Fe and Mn-oxidizing bacteria appear to be successful: sequenced isolates anticipated in the near-term, to be compared to clone libraries. Noble gas & stable-isotope geochemistry will constrain the age & source of the fluids. –Preliminary 14 C data shows 26,000 years