Description: The influence of hydrostatic pressure on microbial sulfate reduction (SR) was studied using sediments obtained at cold seep sites from 5500 to 6200 m water depth of the Japan Trench. Sediment samples were stored under anoxic conditions for 17 months in slurries at 4°C and at in situ pressure (50 MPa), at atmospheric pressure (0.1 MPa), or under methanic conditions with a methane partial pressure of 0.2 MPa. Samples without methane amendment stored at in situ pressure retained higher levels of sulfate reducing activity than samples stored at 0.1 MPa. Piezophilic SR showed distinct substrate specificity after hydrogen and acetate addition. SR activity in samples stored under methanic conditions was one order of magnitude higher than in non-amended samples. Methanic samples stored under low hydrostatic pressure exhibited no increased SR activity at high pressure even with the amendment of methane. These new insights into the effects of pressure on substrate specific sulfate reducing activity in anaerobic environmental samples indicate that hydrostatic pressure must be considered to be a relevant parameter in ecological studies of anaerobic deep-sea microbial processes and long-term storage of environmental samples.

Description: Sediment-hosting hydrothermal systems in the Okinawa Trough maintain a large amount of liquid, supercritical and hydrate phases of CO2 in the seabed. The emission of CO2 may critically impact the geochemical, geophysical and ecological characteristics of the deep-sea sedimentary environment. So far it remains unclear whether microbial communities that have been detected in such high-CO2 and low-pH habitats are metabolically active, and if so, what the biogeochemical and ecological consequences for the environment are. In this study, RNA-based molecular approaches and radioactive tracer-based respiration rate assays were combined to study the density, diversity and metabolic activity of microbial communities in CO2-seep sediment at the Yonaguni Knoll IV hydrothermal field of the southern Okinawa Trough. In general, the number of microbes decreased sharply with increasing sediment depth and CO2 concentration. Phylogenetic analyses of community structure using reverse-transcribed 16S ribosomal RNA showed that the active microbial community became less diverse with increasing sediment depth and CO2 concentration, indicating that microbial activity and community structure are sensitive to CO2 venting. Analyses of RNA-based pyrosequences and catalyzed reporter deposition-fluorescence in situ hybridization data revealed that members of the SEEP-SRB2 group within the Deltaproteobacteria and anaerobic methanotrophic archaea (ANME-2a and -2c) were confined to the top seafloor, and active archaea were not detected in deeper sediments (13–30 cm in depth) characterized by high CO2. Measurement of the potential sulfate reduction rate at pH conditions of 3–9 with and without methane in the headspace indicated that acidophilic sulfate reduction possibly occurs in the presence of methane, even at very low pH of 3. These results suggest that some members of the anaerobic methanotrophs and sulfate reducers can adapt to the CO2-seep sedimentary environment; however, CO2 and pH in the deep-sea sediment were found to severely impact the activity and structure of the microbial community.

Description: The occurrence of microbially induced smectite-to-illite (S-I) reaction has challenged both the notions of solely inorganic chemical control for this reaction and the conventional concept of a semiquantitative illite geothermometer for the reconstruction of the thermal and tectonic histories of sedimentary basins. Here, we present evidence for a naturally occurring microbially induced S-I transition, via biotic reduction of phyllosilicate structural Fe(III), in mudstones buried at the Nankai Trough, offshore Japan (International Ocean Discovery Program Site C0023). Biotic S-I reaction is a consequence of a bacterial survival and growth strategy at diagenetic temperatures up to 80 °C within the Nankai Trough mudstones. These results have considerable implications for petroleum exploration, modification of fault behavior, and the understanding of microbial communities in the deep biosphere.

Description: The occurrence of microbially induced smectite-to-illite (S-I) reaction has challenged both the notions of solely inorganic chemical control for this reaction and the conventional concept of a semiquantitative illite geothermometer for the reconstruction of the thermal and tectonic histories of sedimentary basins. Here, we present evidence for a naturally occurring microbially induced S-I transition, via biotic reduction of phyllosilicate structural Fe(III), in mudstones buried at the Nankai Trough, offshore Japan (International Ocean Discovery Program Site C0023). Biotic S-I reaction is a consequence of a bacterial survival and growth strategy at diagenetic temperatures up to 80 °C within the Nankai Trough mudstones. These results have considerable implications for petroleum exploration, modification of fault behavior, and the understanding of microbial communities in the deep biosphere.

Description: IODP Expedition 370 (Temperature Limit of the Deep Biosphere off Muroto) established Site C0023 down to 1180 mbsf in the Nankai Trough off Shikoku Island, Japan, to explore the upper temperature limit of microbial life in deep subseafloor sediments. Part of the scientific program is to investigate the availability of nutrients and energy substrates and to identify unique geochemical and microbial signatures that differentiate the biotic and abiotic realms and/or their transitions (Heuer et al., 2017). Iron (Fe) reduction is considered one of the most ancient forms of microbial respiration (Vargas et al., 1998). In addition, Fe reducers can grow under high temperature and pressure conditions (Kashefi and Lovley, 2003), suggesting that microbes that use Fe oxides as energy substrates are potential candidates to survive close to the temperature limit of the deep biosphere. In this study, we aim at assessing the role of Fe oxides for microbial respiration and the related diagenetic alterations in deep sediments of Site C0023 by applying sequential extractions of Fe oxide and sulfide minerals. Volcanic ash layers, which are ubiquitous in sediments of Site C0023, are of particular interest as they have been identified earlier as hotspots for microbial life (e.g., Inagaki et al., 2003). Torres et al. (2015) further showed that ash layers at a different site in the Nankai Trough are typically rich in Fe and Mn oxides. Their results support the findings of Treude et al. (2014) who postulate a coupling of microbial processes to mineralogy. In addition, on-board measurements show a release of dissolved Fe into the pore water in the depth interval associated with volcanic ash layers (Heuer et al., 2017), suggesting that the observed liberation of dissolved Fe is related to an alteration of Fe phases in these ash layers. Our results show that the total Fe content in sediments of Site C0023 is relatively constant at ~4.2 wt%. The reactive Fe oxide content represents 25% of the total Fe. Based on sequential extractions, the fraction associated with amorphous Fe oxide such as ferrihydrite and lepidocrocite is the dominant Fe fraction with ~0.7 wt%. Mineralogical analyses are currently conducted in order to determine specific Fe mineral phases within this fraction. The total Fe contents in the ash layer samples strongly vary between 1.4 and 6.8 wt%. However, most samples generally contain less total Fe than the surrounding sediments. Similarly, the contents of the reactive Fe oxides are significantly lower. Thus, reactive Fe oxides in ash layers at Site C0023 do not seem to represent the energy substrate for microbial Fe reduction. As one of the next steps, stable Fe isotope (δ56Fe) analyses will be performed on (1) pore-water samples, the (2) different Fe oxide phases and (3) sediment residues remaining after sequential extractions in order to trace the source and reaction pathway for the observed release of dissolved Fe into the pore water. Diagenetic Fe cycling, in particular the reductive dissolution of Fe oxides driven by the reaction with hydrogen sulfide, may lead to the transformation of reactive Fe oxides to Fe sulfides such as pyrite (e.g., Berner 1970). Fe monosulfide contents are below detection limit in sediments of Site C0023. Pyrite, in contrast, occurs over the whole core interval with strongly varying contents. Three significant peaks with contents up to 0.5 wt% could be observed at 552, 707 and 1033 mbsf. The pyrite profile generally mimics the total sulfur profile, which suggests that most of bulk sulfur is present as pyrite. Fe bound in pyrite (Fepyrite), however, only represents less than 5% of the total Fe pool, except for the interval with elevated pyrite contents where Fepyrite accounts for ~10% of bulk Fe. This indicates that sulfidation does not affect the whole Fe oxide pool in sediments of Site C0023. The reductive dissolution of primary ferrimagnetic Fe oxides and the formation of secondary paramagnetic pyrite is generally known to modify rock magnetic properties such as magnetic susceptibility (e.g., Berner, 1970). Thus, our geochemical results are presented in combination with post-cruise generated magnetic susceptibility data. By combining the geochemical methods, including sequential Fe oxide and sulfide extractions and subsequent δ56Fe analyses, with rock magnetic measurements, we intend to decipher the role of Fe mineral phases in maintaining deep subsurface life at Site C0023. Acknowledgements - This research used samples and data provided by the International Ocean Discovery Program (IODP). We would like to thank all personnel involved in the operations aboard the DV Chikyu during Expedition 370 and the support team at the Kochi Core Center. We further would like to thank the German Research Foundation (DFG) for funding this project (project number: 388260220) in the framework of the priority program 527 (Bereich Infrastruktur – International Ocean Discovery Program). References: Berner, R.A., 1970. Sedimentary pyrite formation. AJS 268: 1-23. Heuer, V.B., Inagaki, F., Morono, Y., Kubo, Y., Maeda, L., and the Expedition 370 Scientists, 2017. Expedition 370 Preliminary Report: Temperature Limit of the Deep Biosphere off Muroto. International Ocean Discovery Program. Inagaki, F., Suzuki, M., Takai, K., Oida, H., Sakamoto, T., Aoki, K., Nealson K.H., Horikoshi, K., 2003. Microbial communities associated with geological horizons in coastal subseafloor sediments from the Sea of Okhotsk. AEM 69: 7224-7235. Kashefi, K., Lovley, D.R., 2003. Extending the upper temperature limit of life. Science 301: 934. Torres, M.E., Cox, T., Hong, W.-L., McManus, J., Sample, J.C., Destrigneville, C., Gan, H.M., Gan, H.Y., Moreau J.W., 2015. Crustal fluid and ash alteration impacts on the biosphere of Shikoku Basin sediments, Nankai Trough, Japan. Geobiology 13: 562-580. Treude, T., Krause, S., Maltby, S., Dale, A.W., Coffin, R., Hamdan, L.J., 2014. Sulfate reduction and methane oxidation activity below the sulfate-methane transition zone in Alaskan Beaufort Sea continental margin sediments: Implications for deep sulfur cycling. GCA 144: 217-237. Vargas, M., Kashefi, K., Blunt-Harris, E.L., Lovley, D.E., 1998. Microbiological evidence for Fe(III) reduction on early Earth. Nature 395: 65-67.

Description: Iron reduction is one of the most ancient forms of microbial respiration. This and the observation that iron reducers can grow under high temperature and pressure conditions suggests that they may play an important role in the deep biosphere. We will use stable Fe isotopes to disentangle microbial and abiotic processes involved in deep Fe cycling at IODP Site C0023 in the Nankai Trough. This will help to reach the goal of Expedition 370: ”T-Limit of the Deep Biosphere off Muroto” – the assessment of how microbial communities change with increasing sediment depth and temperature, by which factors changes are controlled, and where microbial life ceases. Dissolved iron was found at Site C0023 only within the methanic zone from 400 to 600 mbsf. The total drilling depth was 1180 mbsf. Is the Fe2+ release coupled to microbial activity? If yes, is it confined to the 200 m thick interval due to presence of reactive Fe minerals or because the microbes cannot cope with the temperatures prevailing in deeper sediments? Microbial iron reduction is known to cause pronounced enrichments of 54Fe in pore water, which should also be reflected by authigenic Fe minerals. The residual Fe pool, in contrast, becomes progressively enriched in 56Fe. Kinetic reactions of iron with sulfide enrich 56Fe in pore water, which allows a discrimination between microbial reduction and abiotic iron - sulfur interactions based on δ56Fe. As a result of different origins of incorporated Fe and different reactivities towards microbial reduction and sulfidation, Fe minerals in sediments possess different δ56Fe signatures and may show geochemical indications for microbial life. By analyzing δ56Fe of pore water and sequentially leached reactive and refractive Fe phases from Site C0023 sediments we will gain insight into the processes driving Fe2+ liberation at depth and hopefully assess links between the microbial activity and mineralogy (the presence of electron acceptors) as well as temperature.

Description: An International Ocean Discovery Program (IODP) workshop was held at Sydney University, Australia, from 13 to 16 June 2017 and was attended by 97 scientists from 12 countries. The aim of the workshop was to investigate future drilling opportunities in the eastern Indian Ocean, southwestern Pacific Ocean, and the Indian and Pacific sectors of the Southern Ocean. The overlying regional sedimentary strata are underexplored relative to their Northern Hemisphere counterparts, and thus the role of the Southern Hemisphere in past global environmental change is poorly constrained. A total of 23 proposal ideas were discussed, with 12 of these deemed mature enough for active proposal development or awaiting scheduled site survey cruises. Of the remaining 11 proposals, key regions were identified where fundamental hypotheses are testable by drilling, but either site surveys are required or hypotheses need further development. Refinements are anticipated based upon regional IODP drilling in 2017/2018, analysis of recently collected site survey data, and the development of site survey proposals. We hope and expect that this workshop will lead to a new phase of scientific ocean drilling in the Australasian region in the early 2020s.