Investigation of high temperature enhanced hydrogen formation in mineral amended sediment slurries, as a potential novel deep biosphere energy source

The deep biosphere has been estimated to be the largest prokaryotic habitat on Earth. However, what energy sources sustain these prokaryotes at depth and over geological timescales, remote from photosynthetic energy supply, is unclear. Hydrogen generation from basalt weathering has been controversially suggested as a potential deep terrestrial energy source, although other minerals were thought to be unimportant, as they did not generate H2 under abiotic conditions. In this study these experiments were repeated but in the presence of sedimentary prokaryotes using a range of minerals, with differing iron concentrations at a range of temperatures (4-105 C) and under anoxic conditions. Results showed that at certain temperatures high levels of H2 were produced from several minerals (600-1650 umol l"1). This was associated with high levels of acetate formation (-5000-1000 umol l"1). Substantial CH4 (100-600 umol f1) was also produced. Sulphate reduction proceeded between 50-85 C, which no removal at higher temperatures, similar to hot oil reservoirs. The amounts of these compounds produced varied depending on mineral composition. The presence of iron in minerals was found not to be necessary to facilitate the production of H2 as previously thought. Under sterile conditions little H2 or other products were formed. This shows that H2 generation from minerals was a microbially mediated process and was consistent with the presence of considerable prokaryotic cell numbers (range 4.3 x 107 to 5.1 x 108 cells ml"1) in these experiments. Prokaryotic cell numbers decrease with increases in temperature, similar to the predicted decrease in numbers with depth in the sub seafloor biosphere (e.g. 99 C 6.67 x 105 cells ml"1). Sequential ramping experiments to 155 C, demonstrated that prokaryotic activity in the biotic zone 120 C. This has implications for hydrous pyrolysis experiments, which are used to simulate high temperature diagenesis. This increases the sources of potential energy, which may migrate upward to feed the base of the deep biosphere. The prokaryotic community present was assessed using 16S rRNA gene analysis (PCR-DGGE). Communities varied with mineral type and included thermophilic methanogens and acetate oxidisers, iron-reducers and Deltaproteobacteria, Thermoplasmatales, as well as uncultured bacterial and archaeal sequences. Clone libraries indicate that the archaeal community is similar to proposed HyperSliMe communities, dominated by Thermococcales related species at high temperature 80- 95 C. Fresh grinding of minerals enhanced activity, which has implications for the rate of energy supply in the subsurface. A mechanochemical process based on the interaction of Si radicals, which is enhanced by the presence of prokaryotes, perhaps through microbial weathering, is hypothesised to be involved in H2 generation. But the interaction of other mineral constituents such as Fe, Al and Ti may also play a role. We found minerals stimulated prokaryotic activity up to at least 100 days. Interestingly, the carbon isotope values for the CH4 produced covered a wide range of values (-42 to -78‰), including depleted values that could be misinterpreted as being indicative of an abiotic origin. These results extend the range of minerals that could be potential "dark energy" sources in the subsurface and provide details of the prokaryotes and processes that such energy sources could stimulate.