International audienceA general review of the sub-seafloor biosphere is presented. This includes an update and assessment of prokary-otic cell distributions within marine sediments, current deepest 1922 m, and the impact of this on global sub-seafloor biomass estimates. These global estimates appear relatively robust to different calculation approaches and our updated estimate is 5.39 × 10 29 cells, taking into consideration new data from very low organic matter South Pacific Gyre sediments. This is higher than other recent estimates, which is justified as several sediments, such as gas hydrate deposits and oil reservoirs, can have elevated cell concentrations. The proposed relationship between elevated cell concentrations and Milankovitch Cycles in sequential diatom rich layers at some sites, demonstrates not only a dynamic deep biosphere, but also that the deep biosphere is an integral part of Earth System Processes over geological time scales. Cell depth distributions vary in different oceanographic provinces and this is also reflected in contrasting biodiversity. Despite this there are some clear common, sub-seafloor prokary-otes, for Bacteria these are the phyla Chloroflexi, Gammaproteobacteria, Planctomycetes and the candidate phylum JS1, and for Archaea uncultivated lineages within the phylum Crenarchaeota (Miscellaneous Crenarchaeotal Group and Marine Benthic Group B), Euryarchaeota (SAGMEG, Marine Benthic Group-D/Thermoplasmatales associated groups) and Thaumarchaeota (Marine Group I). In addition, spores, viruses and fungi have been detected, but their importance is not yet clear. Consistent with the direct demonstration of active prokaryotic cells, prokaryotes have been enriched and isolated from deep sediments and these reflect a subset of the total diversity, including spore formers that are rarely detected in DNA analyses. Activities are generally low in deep marine sediments (~10,000 times lower than in near-surface sediments), however, depth integrated activity calculations demonstrate that sub-surface sediments can be responsible for the majority of sediment activity (up to 90%), and hence, are biogeochemically important. Unlike near-surface sediments, competitive metabolisms can occur together and metabolism per cell can be 1000 times lower than in culture, and below the lowest known maintenance energies. Consistent with this, cell turnover times approach geological timescales (100–1000s of years). Prokaryotic necromass may be an important energy and carbon source, but this is largely produced in near-surface sediments as cell numbers rapidly decrease. However, this and deposited organic matter may be activated at depth as temperatures increase. At thermogenic temperatures methane and other hydrocarbons, plus H 2 , acetate and CO 2 may be produced and diffuse upwards to feed the base of the biosphere (e.g. Nankai Trough and Newfoundland Margin). Temperature activation of minerals may also result in oxidation of sulphides and the formation of electron acceptors, plus H 2 from low temperature (~55 °C) serpentenisation and water radiolysis. New mineral surface formation from fracturing, weathering and subduction etc. can also mechanochemically split water producing both substrates (H 2) and oxidants (O 2 and H 2 O 2) for prokaryotes. These and other biosphere:geosphere interactions may be important for sustaining a globally significant sub-seafloor biosphere

Sub-sea-floor sediments may contain two-thirds of Earth's total prokaryotic biomass. However, this has its basis in data extrapolation from ~500-meter to 4-kilometer depths, whereas the deepest documented prokaryotes are from only 842 meters. Here, we provide evidence for low concentrations of living prokaryotic cells in the deepest (1626 meters below the sea floor), oldest (111 million years old), and potentially hottest (~100 degrees C) marine sediments investigated. These Newfoundland margin sediments also have DNA sequences related to thermophilic and/or hyperthermophilic Archaea. These form two unique clusters within Pyrococcus and Thermococcus genera, suggesting unknown, uncultured groups are present in deep, hot, marine sediments (~54 degrees to 100 degrees C). Sequences of anaerobic methane-oxidizing Archaea were also present, suggesting a deep biosphere partly supported by methane. These findings demonstrate that the sub-sea-floor biosphere extends to at least 1600 meters below the sea floor and probably deeper, given an upper temperature limit for prokaryotic life of at least 113 degrees C and increasing thermogenic energy supply with depth.

Stable isotope probing of prokaryotic DNA was used to determine active prokaryotes using 13C-labelled substrates (glucose, acetate, CO2) in sediment s

Nine marine methanogenic Methanococcoides strains, including the type strains of Methanococcoides methylutens , M. burtonii , and M. alaskense , were tested for the utilization of N -methylated glycines. Three strains (NM1, PM2, and MKM1) used glycine betaine ( N , N , N -trimethylglycine) as a substrate for methanogenesis, partially demethylating it to N , N -dimethylglycine, whereas none of the strains used N , N -dimethylglycine or sarcosine ( N -methylglycine). Growth rates and growth yields per mole of substrate with glycine betaine (3.96 g [dry weight] per mol) were similar to those with trimethylamine (4.11 g [dry weight] per mol). However, as glycine betaine is only partially demethylated, the yield per methyl group was significantly higher than with trimethylamine. If glycine betaine and trimethylamine are provided together, trimethylamine is demethylated to dimethyl- and methylamine with limited glycine betaine utilization. After trimethylamine is depleted, dimethylamine and glycine betaine are consumed rapidly, before methylamine. Glycine betaine extends the range of substrates that can be directly utilized by some methanogens, allowing them to gain energy from the substrate without the need for syntrophic partners.

Bacterial spores are widespread in marine sediments, including those of thermophilic, sulphate-reducing bacteria, which have a high minimum growth temperature making it unlikely that they grow in situ. These Desulfotomaculum spp. are thought to be from hot environments and are distributed by ocean currents. Their cells and spores upper temperature limit for survival is unknown, as is whether they can survive repeated high-temperature exposure that might occur in hydrothermal systems. This was investigated by incubating estuarine sediments significantly above (40–80 °C) maximum in situ temperatures (∼23 °C), and with and without prior triple autoclaving. Sulphate reduction occurred at 40–60 °C and at 60 °C was unaffected by autoclaving. Desulfotomaculum sp. C1A60 was isolated and was most closely related to the thermophilic D. kuznetsovii^T (∼96% 16S rRNA gene sequence identity). Cultures of Desulfotomaculum sp. C1A60, D. kuznetsovii^Tand D. geothermicum B2T survived triple autoclaving while other related Desulfotomaculum spp. did not, although they did survive pasteurisation. Desulfotomaculum sp. C1A60 and D. kuznetsovii cultures also survived more extreme autoclaving (C1A60, 130 °C for 15 min; D. kuznetsovii, 135 °C for 15 min, maximum of 154 °C reached) and high-temperature conditions in an oil bath (C1A60, 130° for 30 min, D. kuznetsovii 140 °C for 15 min). Desulfotomaculum sp. C1A60 with either spores or predominantly vegetative cells demonstrated that surviving triple autoclaving was due to spores. Spores also had very high culturability compared with vegetative cells (∼30 × higher). Combined extreme temperature survival and high culturability of some thermophilic Desulfotomaculum spp. make them very effective colonisers of hot environments, which is consistent with their presence in subsurface geothermal waters and petroleum reservoirs.