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We compiled DNA and RNA isolation protocols for sediment bulk extraction and their yields from Guaymas Basin subsurface sediments recovered during International Ocean Discovery Program Expedition 385 and evaluated their sensitivity for metagenomic and amplicon analyses of subsurface microbial communities. Guaymas Basin sediments present a challenge for DNA and RNA recovery due to high concentrations of hydrocarbons, steep thermal gradients, and rapidly declining cell numbers downhole. Metagenomic library construction and sequencing was possible from as little as 0.2 to 0.5 ng DNA/cm3 sediment; polymerase chain reaction (PCR) amplification of 16S rRNA genes required in most cases approximately 1–2 ng DNA/cm3 sediment. At in situ temperatures of 50° to 60°C, decreasing DNA recovery leads to increasingly uncertain hit or miss outcomes and failures for metagenomic and amplicon analyses. DNA concentration profiles show that, even before these hot temperatures are reached, relatively moderate temperatures (near 40°C) have a major effect on microbial abundance and DNA yield. Comparison with cell count profiles shows that hydrothermal influence reduces downhole cell densities by multiple orders of magnitude compared to nonhydrothermal sediments. This effect is also visible at relatively moderate temperatures. RNA recovery is highly sensitive to downhole increasing temperatures and decreasing cell numbers, and was most efficient for microbial communities in cool, relatively shallow subsurface sediments. 

Previous studies of microbial communities in subseafloor sediments reported that microbial abundance and diversity decrease with sediment depth and age, and microbes dominating at depth tend to be a subset of the local seafloor community. However, the existence of geographically widespread, subsurface-adapted specialists is also possible. Here, we usemetagenomic and metatranscriptomic analyses of the hydrothermally heated, sediment layers of Guaymas Basin (Gulf of California, Mexico) to examine the distribution and activity patterns of bacteria and archaea along thermal, geochemical and cell count gradients. We find that the composition and distribution of metagenome-assembled genomes (MAGs), dominated by numerous lineages of Chloroflexota and Thermoproteota, correlate with biogeochemical parameters as long as temperatures remain moderate, but downcore increasing temperatures beyond ca. 45 ºC override other factors. Consistently, MAG size and diversity decrease with increasing temperature, indicating a downcore winnowing of the subsurface biosphere. By contrast, specific archaeal MAGs within the Thermoproteota and Hadarchaeota increase in relative abundance and in recruitment of transcriptome reads towards deeper, hotter sediments, marking the transition towards a specialized deep, hot biosphere. 

Analyses of gene expression of subsurface bacteria and archaea provide insights into their physiological adaptations to in situ subsurface conditions. We examined patterns of expressed genes in hydrothermally heated subseafloor sediments with distinct geochemical and thermal regimes in Guaymas Basin, Gulf of California, Mexico. RNA recovery and cell counts declined with sediment depth, however, we obtained metatranscriptomes from eight sites at depths spanning between 0.8 and 101.9m below seafloor. We describe the metabolic potential of sediment microorganisms, and discuss expressed genes involved in tRNA, mRNA, and rRNA modifications that enable physiological flexibility of bacteria and archaea in the hydrothermal subsurface. Microbial taxa in hydrothermally influenced settings like Guaymas Basin may particularly depend on these catalytic RNA functions since they modulate the activity of cells under elevated temperatures and steep geochemical gradients. Expressed genes for DNA repair, protein maintenance and circadian rhythm were also identified. The concerted interaction of many of these genes may be crucial for microorganisms to survive and to thrive in the Guaymas Basin subsurface biosphere. 

The extremely large slip that occurred on the shallow portion of the Japan Trench subduction zone during the 2011 Mw 9.1 Tohoku-oki earthquake directly contributed to the devastating tsunami that inundated the Pacific coast of Japan. International Ocean Discovery Program (IODP) Expedition 405 (Tracking Tsunamigenic Slip Across the Japan Trench) aimed to investigate the conditions and processes that facilitated the extremely shallow slip on the subduction interface during the 2011 Tohoku-oki earthquake to improve understanding of the factors that allow slip to the trench on subduction zones. Expedition 405 implemented a combined logging, coring, and observatory operational plan at two sites: Site C0026, ~8 km seaward of the Japan Trench, to characterize the input sediments to the subduction zone and Site C0019, ~6 km landward of the trench, where the plate boundary fault zone is present at ~825 meters below seafloor (mbsf). At Site C0026, the input section was logged to ~430 mbsf with a logging-while-drilling (LWD) assembly that characterized the succession of sediments and rocks from the seafloor to the basaltic rocks of the oceanic crust. Cores recovered from four holes as deep as 290 mbsf contain a sequence of hemipelagic and pelagic sediments that will be input into the shallow subduction system and therefore control both the localization of the plate boundary fault zone and the slip behavior of the plate boundary. Site C0019 was previously drilled in 2012 during Integrated Ocean Drilling Program Expedition 343 (Japan Trench Fast Drilling Project [JFAST]), and revisiting this site allowed temporal variations in the frontal prism and plate boundary fault zone to be evaluated. The LWD data to ~980 mbsf characterized the frontal prism, plate boundary fault zone, and lower plate to the basaltic volcanic rocks. Cores were recovered from multiple holes that contain a variety of muds from the frontal prism and the plate boundary fault zone, as well as lower plate materials. Comparison with the sediments from Site C0026 provides a basis to interpret the tectonic and sedimentological processes operating in the dynamic environment of the frontal prism. Cores from the plate boundary fault zone provide a unique window into the structural complexity of an active plate boundary fault that is known to host large seismic slip. Two borehole observatories were installed at Site C0019 that contain temperature sensors deployed to take measurements over a period of years and reveal the hydrogeologic structure of the shallow subduction system. These hugely successful drilling operations, combined with postexpedition work to measure the mechanical, frictional, paleomagnetic, and hydrogeologic properties of the core samples and to constrain the history of past seismic slip at Site C0019, will provide an unprecedented opportunity to advance our understanding of shallow subduction systems. Outreach during the expedition leveraged and elevated the success of the operations by sharing the outcomes with a variety of domestic and international audiences, including scientists, students, educators, stakeholders, and the general public. Thanks to the efforts of a large group of onboard outreach officers and their onshore support, activities included ship-to-shore broadcast events; interviews with science party members and crew; the publication of videos, blogs, magazine articles, and social media posts; and development of formalized classroom lesson plans and materials. 

International Ocean Discovery Program (IODP) Expedition 370 explored the limits of the biosphere in the deep subseafloor where temperature exceeds the known temperature maximum of microbial life (~120°C) at the sediment/basement interface ~1.2 km below the seafloor. Site C0023 is located in the protothrust zone in the Nankai Trough off Cape Muroto at a water depth of 4776 m, in the vicinity of Ocean Drilling Program (ODP) Sites 808 and 1174. In 2000, ODP Leg 190 revealed the presence of microbial cells at Site 1174 to a depth of ~600 meters below seafloor (mbsf), which corresponds to an estimated temperature of ~70°C, and reliably identified a single zone of elevated cell concentrations just above the décollement at around 800 mbsf, where temperature presumably reached 90°C; no cell count data was reported for other sediment layers in the 70°–120°C range because the detection limit of manual cell counting for low-biomass samples was not low enough. With the establishment of Site C0023, we aimed to detect and investigate the presence or absence of life and biological processes at the biotic–abiotic transition utilizing unprecedented analytical sensitivity and precision. Expedition 370 was the first expedition dedicated to subseafloor microbiology that achieved time-critical processing and analyses of deep biosphere samples, conducting simultaneous shipboard and shore-based investigations. Our primary objective during Expedition 370 was to study the relationship between the deep subseafloor biosphere and temperature. We comprehensively studied the controls on biomass, activity, and diversity of microbial communities in a subseafloor environment where temperatures increase from ~2°C at the seafloor to ~120°C and thus likely encompasses the biotic–abiotic transition zone. This included investigating whether the supply of fluids containing thermogenic and/or geogenic nutrient and energy substrates may support subseafloor microbial communities in the Nankai accretionary complex. To address these primary scientific objectives and questions, we penetrated 1180 m and recovered 112 cores of sediment and across the sediment/basalt interface. More than 13,000 samples were collected, and selected samples were transferred to the Kochi Core Center by helicopter for simultaneous microbiological sampling and analysis in laboratories with a super-clean environment. In addition to microbiological measurements, we determined the geochemical, geophysical, and hydrogeological characteristics of the sediment and the underlying basement and installed a 13-thermistor sensor borehole temperature observatory into the borehole at Site C0023.