GEOLOGY, March 2011 219 INTRODUCTION The large estimates of prokaryotic biomass in the subseafloor biosphere (Whitman et al., 1998) have been questioned on the basis of insufficient energy supply (D’Hondt et al., 2002). However, increases in temperatures during burial may pro- vide energy by making recalcitrant organic mat- ter more degradable for prokaryotes (Wellsbury et al., 1997). In addition, products of thermo- genic organic matter alteration (>100 °C) can diffuse upward to feed the base of the deep bio- sphere (Roussel et al., 2008; Parkes et al., 1994). However, the suggestion that H 2 from oxidation of ferrous iron minerals by water at low temper- atures might be an additional significant energy source for the deep biosphere (Stevens and McKinley, 1995) has been challenged (Ander- son et al., 1998), as mineral:sterile groundwater incubations produce limited and only transitory amounts of H 2 . Nevertheless, in deep sediments the combined effects of prokaryotic activity, presence of organic matter, and increasing tem- peratures on mineral:water H 2 generation from a range of minerals would occur, but this has not yet been investigated. Here we report the results of such studies and the direct impact of prokary- otic activity on thermogenic processes at even higher temperatures. METHODS Natural rock minerals (X-ray diffraction and inductively coupled plasma-optical emission spectrometry were used to confirm mineral type and chemical composition; see Table DR2 in the GSA Data Repository 1 ) were ground (agate centrifugal ball mill) and added (100 g/L) to 25% Tamar Estuary sediment (St. John’s Lake, UK) anoxic slurries (Parkes et al., 2007) plus ~1% Guaymas Basin (Jorgensen et al., 1992) sediment inoculum. These were incubated (unshaken and in the dark) in 7 mL crimp vials for thermal gradient experiments (Wellsbury et al., 1997) or in a high-pressure vessel (Parr Instrument Company). Headspace gases were analyzed by a natural gas analyzer (PerkinElmer Clarus 500) or gas chromatography combustion isotope ratio mass spectrometer (Varian 3400GC with ThermoElectron XP mass spectrometer) for stable carbon isotopes and pore water by ion chromatography (Dionex ICS-2000). Gas con- centrations are presented as experiment head- space (in μmol/L). Sterile control slurries were triple autoclaved to ensure long-term sterility. RockEval analysis was conducted as described by Parkes et al. (2007). DNA was extracted from slurries using the FastDNA Spin Kit For Soil (MP Biomedicals) and stored at -80 °C. Polymerase chain reac- tion (PCR) was conducted with 16S rRNA gene primers for Bacteria (27F-907R) and Archaea (109F-958R or 109F-PARCH519R) as described by Webster et al. (2006). Products were reamplified by nested PCR (Bacteria, 357F-518R; Archaea, SAF-PARCH519R) and analyzed by density gradient gel electrophore- sis (DGGE) with a 30%–60% denaturant gra- dient. Archaeal products were also analyzed on a 30%–80% denaturant gradient due to the higher guanine-cytosine content of thermophilic archaeal sequences. Selected DGGE bands were excised and sequenced using an Applied Bio- systems 3130xl Genetic Analyzer. In addition, for some samples (ilmenite and pyrite slurries incubated at 63–90 °C) replicate initial PCR products were pooled and cloned using the pGEM-T Easy Vector System (Pro- mega). We randomly chose 16S rRNA gene clones from each library (25) and sequenced them. Sequences were checked for chimeras and assigned to phylogenetic groups by com- parison with the National Center for Biotech- nology Information database (http://www.ncbi. nlm.nih.gov/); assignment was confirmed by phylogenetic tree reconstruction with neighbor- joining and Jukes and Cantor correction and Minimum Evolution and LogDet in Molecular Evolutionary Genetics Analysis Software Ver- sion 4.0. All sequences have been submitted to the European Molecular Biology Laboratory nucleotide sequence database under accession numbers FR692121–FR692180. RESULTS AND DISCUSSION Temperature gradient incubation experiments were conducted (~0–100 °C, to 130 days) with a range of minerals and rock, both iron and non-iron (hematite, labradorite, pyrite, basalt, ilmenite, hornblende, olivine, magnetite, quartz sand), added to the sediment slurries, which pro- vided both a prokaryotic inoculum and a natu- ral mineral and organic matter matrix. Mineral additions resulted in considerable H 2 (maximum 1626 μM) and acetate production (maximum ~10,000 μM), which increased with incubation time and temperature (e.g., basalt, Fig. 1A; 83 days incubation). During incubation some H 2 and acetate were probably also consumed to fuel the stimulated sulfate removal and CH 4 produc- tion (maximum 536 μM), as both are important substrates for sulfate reduction and methanogen- esis (Parkes et al., 1989; Whiticar et al., 1986). These reactions were mediated by prokaryotes, as sterile controls had negligible H 2 and CH 4 for- mation and sulfate removal, even at temperatures Geology, March 2011; v. 39; no. 3; p. 219–222; doi: 10.1130/G31598.1; 2 figures; 1 table; Data Repository item 2011086. © 2011 Geological Society of America. For permission to copy, contact Copyright Permissions, GSA, or editing@geosociety.org. *E-mail: ParkesRJ@cf.ac.uk. Prokaryotes stimulate mineral H 2 formation for the deep biosphere and subsequent thermogenic activity R. John Parkes 1 *, Cathal D. Linnane 1 , Gordon Webster 1 , Henrik Sass 1 , Andrew J. Weightman 2 , Ed R.C. Hornibrook 3 , and Brian Horsfield 4 1 School of Earth and Ocean Sciences, Cardiff University, Main Building, Cardiff, Wales CF10 3AT, UK 2 Cardiff School of Biosciences, Cardiff University, Main Building, Cardiff, Wales, CF10 3AT, UK 3 Department of Earth Sciences, University of Bristol, Wills Memorial Building, Bristol, BS8 1RJ, UK 4 GeoForschungsZentrum (GFZ), Telegrafenberg, B425 D-14473 Potsdam, Germany 1 GSA Data Repository item 2011086, Tables DR1-DR3, Figures DR1-DR8, and references, is available online at www.geosociety.org/pubs/ft2011 .htm, or on request from editing@geosociety.org or Documents Secretary, GSA, P.O. Box 9140, Boulder, CO 80301, USA. ABSTRACT The deep subseafloor biosphere contains two-thirds of Earth’s prokaryotic biomass, which may indicate the presence of novel mechanisms of energy generation as temperatures increase in the subsurface. In sediment slurry experiments (0–100 °C) with a range of common min- erals and rocks (including basalt and quartz), there is significant H 2 formation at elevated temperatures, but only in the presence of prokaryotes. This stimulates further prokaryotic activity, typical of deep sediments (sulfate reduction, acetogenesis, and CO 2 production, plus continuing methanogenesis), and Bacteria and Archaea representative of many deep sediment types develop. H 2 and acetate formation is particularly stimulated above 70 °C. This pro- karyotic activity even enhances reactions when temperatures are raised to thermogenic levels (~125–155 °C), including hydrocarbon generation. Mechanochemistry may be important for mineral H 2 formation; this is enhanced by prokaryotes (biomechanochemistry), and subsur- face stress and fracturing, which is widespread on Earth. on June 5, 2015 geology.gsapubs.org Downloaded from