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Abstract Environmental factors shape subsurface microbial ecosystems, and well-characterized sites are ideal for determining how environmental parameters shape sediment communities. Sediments from eight geologically and thermally distinct sites were drilled during International Ocean Discovery Program Expedition 385 in Guaymas Basin, an expedition focused on the hydrothermal deep biosphere. Using high-throughput 16S ribosomal nucleic acid sequencing, cell counts, phylogenetics, metatranscriptomics, and mineralogical/elemental X-ray spectroscopy, we examine linkages and feedbacks between mineral composition, temperature, geochemistry, and microbial populations. We show subsurface life is dominated by heterotrophic, cosmopolitan prokaryotes that thrive within a range of sediments and temperature conditions across Guaymas Basin. Hydrothermally-affiliated lineages are detected in low numbers at sites with steepest temperature gradients, within communities of mesophilic taxa that occur throughout Guaymas Basin and in other marine subsurface habitats. Thus, hydrothermal lineages do not replace the cosmopolitan, mesophilic subsurface community, but remain specific to sites where volcanic intrusions drive hydrothermal circulation.

The search for life beyond the Earth is the overarching goal of the NASAAstrobiology Program, and it underpins the science of missions that explore theenvironments of Solar System planets and exoplanets. However, the detection ofextraterrestrial life, in our Solar System and beyond, is sufficientlychallenging that it is likely that multiple measurements and approaches,spanning disciplines and missions, will be needed to make a convincing claim.Life detection will therefore not be an instantaneous process, and it isunlikely to be unambiguous-yet it is a high-stakes scientific achievement thatwill garner an enormous amount of public interest. Current and upcomingresearch efforts and missions aimed at detecting past and extant life could besupported by a consensus framework to plan for, assess and discuss lifedetection claims (c.f. Green et al., 2021). Such a framework could helpincrease the robustness of biosignature detection and interpretation, andimprove communication with the scientific community and the public. In responseto this need, and the call to the community to develop a confidence scale forstandards of evidence for biosignature detection (Green et al., 2021), acommunity-organized workshop was held on July 19-22, 2021. The meeting wasdesigned in a fully virtual (flipped) format. Preparatory materials includingreadings, instructional videos and activities were made available prior to theworkshop, allowing the workshop schedule to be fully dedicated to activecommunity discussion and prompted writing sessions. To maximize globalinteraction, the discussion components of the workshop were held duringbusiness hours in three different time zones, Asia/Pacific, European and US,with daily information hand-off between group organizers.

Abstract 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 use metagenomic 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.

A fourth of the global seabed sediment volume is buried at depths where temperatures exceed 80 °C, a previously proposed thermal barrier for life in the subsurface. Here, we demonstrate, utilizing an extensive suite of radiotracer experiments, the prevalence of active methanogenic and sulfate-reducing populations in deeply buried marine sediment from the Nankai Trough subduction zone, heated to extreme temperature (up to ~120 °C). The small microbial community subsisted with high potential cell-specific rates of energy metabolism, which approach the rates of active surface sediments and laboratory cultures. Our discovery is in stark contrast to the extremely low metabolic rates otherwise observed in the deep subseafloor. As cells appear to invest most of their energy to repair thermal cell damage in the hot sediment, they are forced to balance delicately between subsistence near the upper temperature limit for life and a rich supply of substrates and energy from thermally driven reactions of the sedimentary organic matter.

Abstract Dissimilatory iron reduction (DIR) is suggested to be one of the earliest forms of microbial respiration. It plays an important role in the biogeochemical cycling of iron in modern and ancient sediments. Since microbial iron cycling is typically accompanied by iron isotope fractionation, stable iron isotopes are used as tracer for biological activity. Here we present iron isotope data for dissolved and sequentially extracted sedimentary iron pools from deep and hot subseafloor sediments retrieved in the Nankai Trough off Japan. Dissolved iron (Fe(II)aq) is isotopically light throughout the ferruginous sediment interval but some samples have exceptionally light isotope values. Such light values have never been reported in natural marine environments and cannot be solely attributed to DIR. We show that the light isotope values are best explained by a Rayleigh distillation model where Fe(II)aq is continuously removed from the pore water by adsorption onto iron (oxyhydr)oxide surfaces. While the microbially mediated Fe(II)aq release has ceased due to an increase in temperature beyond the threshold of mesophilic microorganisms, the abiotic adsorptive Fe(II)aq removal continued, leading to uniquely light isotope values. These findings have important implications for the interpretation of dissolved iron isotope data especially in deep subseafloor sediments.

Abstract Microbes in marine sediments detected and counted by direct observation of membrane-filtered sediment samples stained with acridine orange. This technique can still be applied to high-biomass (> 105 cells/cm3) sedimentary habitats, such as organic-rich sediments collected in shallow areas near the seafloor. However, to further explore the nutrients and energy turnover under extremely low energy flux conditions, or in habitats that are close to the lower limit of the biosphere, technological breakthroughs have been required to increase the detection sensitivity for microbial life at densities of a few cells/cm3 of sediment. These technological developments contributed to increasing fundamental information on microbial life at the fringes of the subseafloor biosphere and led to the discovery of revivable microbes in sediments aged up to 101.5 million years old. More recently, chemical detection methods have revealed the existence of spores in the deep biosphere that are impermeable to conventional DNA stains. Previous applications of molecular biology-based approaches have been limited to relatively higher biomass samples, potentially because the cells surviving in these very low energy flux environments have less integrated genomes. Here, I review the contribution and importance of the technological developments that have been made in the study of microbes from the subseafloor biosphere, recent developments of alternative methods to microscopically detect microbial spores and their application to deep subseafloor sediments, and the challenges associated with applying molecular biological approaches to study low-biomass samples.

Abstract It is necessary to purify diatom frustules by taxon to perform accurate geochemical analyses of diatom fossils preserved in sediments. However, the small size of diatoms has hitherto prevented taxon-specific purification; therefore, previous geochemical analyses of diatom frustules have been performed with mixtures of various taxa. In this study, we developed a taxon-selective collection method of diatom fossils that uses a cell sorter. The experimental material comprised six samples from a sediment core of Hole U1538A in the Scotia Sea, Atlantic sector of the Southern Ocean drilled during the International Ocean Discovery Program Expedition 382. Following conventional pretreatments (removal of organic matter and carbonates as well as clay minerals), we conducted cell sorter experiments focusing on the optical and fluorescence characteristics of the diatom fossils. We succeeded in selectively isolating with high purity five diatom taxa representing the Southern Ocean diatom flora: (1) discoid diatoms (mainly Thalassiosira) with a moderate degree of fluorescence; (2) Fragilariopsis (mainly Fragilariopsis kerguelensis), which exhibits the highest fluorescence values; (3) Rhizosolenia; (4) Eucampia antarctica; and (5) needle-shaped diatoms (Thalassiothrix). This taxon-specific diatom purification method will enable more accurate geochemical analyses, such as the oxygen isotope ratio (δ18O) of diatom frustules, which is likely to lead to significant advances in paleoceanography, especially at high latitudes or in upwelling zones where diatoms are abundant. This method will also be useful in paleolimnology, ocean biology, and phycology.

Microorganisms in marine subsurface sediments substantially contribute to global biomass. Sediments warmer than 40°C account for roughly half the marine sediment volume, but the processes mediated by microbial populations in these hard-to-access environments are poorly understood. We investigated microbial life in up to 1.2-kilometer-deep and up to 120°C hot sediments in the Nankai Trough subduction zone. Above 45°C, concentrations of vegetative cells drop two orders of magnitude and endospores become more than 6000 times more abundant than vegetative cells. Methane is biologically produced and oxidized until sediments reach 80° to 85°C. In 100° to 120°C sediments, isotopic evidence and increased cell concentrations demonstrate the activity of acetate-degrading hyperthermophiles. Above 45°C, populated zones alternate with zones up to 192 meters thick where microbes were undetectable.

We developed a nondestructive three-dimensional microbial visualization method utilizing synchrotron radiation X-ray microscale computed tomography to better understand the relationship between microorganisms and their surrounding habitats. The method was tested and optimized using a mixture of axenic Escherichia coli and Comamonas testosteroni. The osmium-thiocarbohydrazide-osmium method was used to stain all the microbial cells, and gold in situ hybridization was used to detect specific phylogenetic microbial groups. The stained samples were embedded in epoxy resin for microtomographic analysis. Differences in X-ray absorbances were calculated by subtracting the pre-L3-edge images from the post-L3-edge images to visualize the osmium and gold signals. Although we successfully detected cells stained with osmium, those labeled with gold were not detected, probably because of the insufficient density of gold atoms in the microbial cells. We then applied the developed technique to anaerobic granules and visualized the distribution of microbial cells and extracellular polymeric substances. Empty spaces were highlighted to determine the cavity distribution in granules. Numerous independent cavities of different sizes were identified in the granules. The developed method can be applied to various environmental samples for deeper insights into microbial life in their habitats. [ABSTRACT FROM AUTHOR]

Guaymas Basin, located in the Gulf of California, is a hydrothermally active marginal basin. Due to steep geothermal gradients and localized heating by sill intrusions, microbial substrates like short-chain fatty acids and hydrocarbons are abiotically produced from sedimentary organic matter at comparatively shallow depths. We analyzed the effect of hydrocarbons on uptake of hydrocarbons by microorganisms via nano-scale secondary ion mass spectrometry (NanoSIMS) and microbial sulfate reduction rates (SRR), using samples from two drill sites sampled by IODP Expedition 385 (U1545C and U1546D). These sites are in close proximity of each other (ca. 1 km) and have very similar sedimentology. Site U1546D experienced the intrusion of a sill that has since then thermally equilibrated with the surrounding sediment. Both sites currently have an identical geothermal gradient, despite their different thermal history. The localized heating by the sill led to thermal cracking of sedimentary organic matter and formation of potentially bioavailable organic substrates. There were low levels of hydrocarbon and nitrogen uptake in some samples from both sites, mostly in surficial samples. Hydrocarbon and methane additions stimulated SRR in near-seafloor samples from Site U1545C, while samples from Site U1546D reacted positively only on methane. Our data indicate the potential of microorganisms to metabolize hydrocarbons even in the deep subsurface of Guaymas Basin. [ABSTRACT FROM AUTHOR]

The marine subsurface is a long-term sink of atmospheric carbon dioxide with significant implications for climate on geologic timescales. Subsurface microbial cells can either enhance or reduce carbon sequestration in the subsurface, depending on their metabolic lifestyle. However, the activity of subsurface microbes is rarely measured. Here, we used nanoscale secondary ion mass spectrometry (nanoSIMS) to quantify anabolic activity in 3,203 individual cells from the thermally altered deep subsurface in the Guaymas Basin, Mexico (3-75 m below the seafloor, 0-14°C). We observed that a large majority of cells were active (83%-100%), although the rates of biomass generation were low, suggesting cellular maintenance rather than doubling. Mean single-cell activity decreased with increasing sediment depth and temperature and was most strongly correlated with porewater sulfate concentrations. Intracommunity heterogeneity in microbial activity decreased with increasing sediment depth and age. Using a dual-isotope labeling approach, we determined that all active cells analyzed were heterotrophic, deriving the majority of their cellular carbon from organic sources. However, we also detected inorganic carbon assimilation in these heterotrophic cells, likely via processes such as anaplerosis, and determined that inorganic carbon contributes at least 5% of the total biomass carbon in heterotrophs in this community. Our results demonstrate that the deep marine biosphere at Guaymas Basin is largely active and contributes to subsurface carbon cycling primarily by not only assimilating organic carbon but also fixing inorganic carbon. Heterotrophic assimilation of inorganic carbon may be a small yet significant and widespread underappreciated source of labile carbon in the global subsurface. [ABSTRACT FROM AUTHOR]

The origin of eukaryotes remains unclear.sup.1-4. Current data suggest that eukaryotes may have emerged from an archaeal lineage known as 'Asgard' archaea.sup.5,6. Despite the eukaryote-like genomic features that are found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear, owing to the lack of cultured representatives and corresponding physiological insights. Here we report the decade-long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment. The archaeon--'Candidatus Prometheoarchaeum syntrophicum' strain MK-D1--is an anaerobic, extremely slow-growing, small coccus (around 550 nm in diameter) that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexes have been proposed for Asgard archaea.sup.6, the isolate has no visible organelle-like structure. Instead, Ca. P. syntrophicum is morphologically complex and has unique protrusions that are long and often branching. On the basis of the available data obtained from cultivation and genomics, and reasoned interpretations of the existing literature, we propose a hypothetical model for eukaryogenesis, termed the entangle-engulf-endogenize (also known as E.sup.3) model. Isolation and characterization of an archaeon that is most closely related to eukaryotes reveals insights into how eukaryotes may have evolved from prokaryotes., Author(s): Hiroyuki Imachi [sup.1] , Masaru K. Nobu [sup.2] , Nozomi Nakahara [sup.1] [sup.2] [sup.3] , Yuki Morono [sup.4] , Miyuki Ogawara [sup.1] , Yoshihiro Takaki [sup.1] , Yoshinori Takano [...]

Abstract Biogeochemical processes in subseafloor sediments are closely coupled to global element cycles. To improve the understanding of changes in biogeochemical conditions on geological timescales, we investigate sediment cores from a 1,180 m deep hole in the Nankai Trough offshore Japan (Site C0023) drilled during International Ocean Discovery Program Expedition 370. During its tectonic migration from the Shikoku Basin to the Nankai Trough over the past 15 Ma, Site C0023 has experienced significant changes in depositional, thermal, and geochemical conditions. By combining pore‐water, solid‐phase, and rock magnetic data, we demonstrate that a transition from organic carbon‐starved conditions with predominantly aerobic respiration to an elevated carbon burial environment with increased sedimentation occurred at ∼2.5 Ma. Higher rates of organic carbon burial in consequence of increased nutrient supply and productivity likely stimulated the onset of anaerobic electron‐accepting processes during organic carbon degradation. A significant temperature increase by ∼50°C across the sediment column associated with trench‐style sedimentation since ∼0.5 Ma could increase the bioavailability of organic matter and enhance biogenic methanogenesis. The resulting shifts in reaction fronts led to diagenetic transformation of iron (oxyhydr)oxides into pyrite in the organic carbon‐starved sediments several millions of years after burial. We also show that high amounts of reducible iron(III) which can serve as electron acceptor for microbial iron(III) reduction are preserved and still available as phyllosilicate‐bound iron. This is the first study that shows the evolution of long‐term variations of (bio‐)geochemical processes along tectonic migration of ocean floor, thereby altering the primary sediment composition long after deposition.

High-pressure (HP) environments represent the largest volumetric majority of habitable space for microorganisms on the planet, including the deep-sea and subsurface biosphere. However, the importance of pressure as an environmental variable affecting deep microbial life and their biogeochemical functions in carbon cycling still remains poorly understood. Here, we designed a new high-volume HP-sediment core sampler that is deployable on the payload of a remotely operated vehicle and can maintain in situ HP conditions throughout multi-month enrichment incubations including daily amendments with liquid media and gases and daily effluent sampling for geochemical or microbiological analysis. Using the HP core device, we incubated sediment and overlying water associated with methane hydrate-exposed on the seafloor of the Joetsu Knoll, Japan, at 10 MPa and 4°C for 45 days in the laboratory. Diversity analyses based on 16S rRNA and methane-related functional genes, as well as carbon isotopic analysis of methane and bicarbonate, indicated the stimulation of both aerobic and anaerobic methanotrophy driven by members of the Methylococcales, and ANME, respectively: i.e., aerobic methanotrophy was observed upon addition of oxygen whereas anaerobic processes subsequently occurred after oxygen consumption. These laboratory-measured rates at 10 MPa were generally in agreement with previously reported rates of methane oxidation in other oceanographic locations.