The Lost City hydrothermal field is an iconic example of a microbial ecosystem fueled by energy and carbon from Earth’s mantle. Uplift of mantle rocks into the seafloor can trigger a process known as serpentinization that releases molecular hydrogen (H 2 ) and creates unusual environmental conditions where simple organic carbon molecules are more stable than dissolved inorganic carbon.

The Atlantis Massif rises 4,000 m above the seafloor near the Mid-Atlantic Ridge and consists of rocks uplifted from Earth’s lower crust and upper mantle. Exposure of the mantle rocks to seawater leads to their alteration into serpentinites. These aqueous geochemical reactions, collectively known as the process of serpentinization, are exothermic and are associated with the release of hydrogen gas (H2), methane (CH4), and small organic molecules. The biological consequences of this flux of energy and organic compounds from the Atlantis Massif were explored by International Ocean Discovery Program (IODP) Expedition 357, which used seabed drills to collect continuous sequences of shallow (<16 m below seafloor) marine serpentinites and mafic assemblages. Here, we report the census of microbial diversity in samples of the drill cores, as measured by environmental 16S rRNA gene amplicon sequencing. The problem of contamination of subsurface samples was a primary concern during all stages of this project, starting from the initial study design, continuing to the collection of samples from the seafloor, handling the samples shipboard and in the lab, preparing the samples for DNA extraction, and analyzing the DNA sequence data. To distinguish endemic microbial taxa of serpentinite subsurface rocks from seawater residents and other potential contaminants, the distributions of individual 16S rRNA gene sequences among all samples were evaluated, taking into consideration both presence/absence and relative abundances. Our results highlight a few candidate residents of the shallow serpentinite subsurface, including uncultured representatives of the Thermoplasmata, Acidobacteria, Acidimicrobia, and Chloroflexi. IMPORTANCE The International Ocean Discovery Program Expedition 357—“Serpentinization and Life”—utilized seabed drills to collect rocks from the oceanic crust. The recovered rock cores represent the shallow serpentinite subsurface of the Atlantis Massif, where reactions between uplifted mantle rocks and water, collectively known as serpentinization, produce environmental conditions that can stimulate biological activity and are thought to be analogous to environments that were prevalent on the early Earth and perhaps other planets. The methodology and results of this project have implications for life detection experiments, including sample return missions, and provide a window into the diversity of microbial communities inhabiting subseafloor serpentinites.

The Atlantis Massif rises 4,000 m above the seafloor near the Mid-Atlantic Ridge and consists of rocks uplifted from Earth's upper mantle. Exposure of the mantle rocks to seawater leads to their alteration into serpentinites. These aqueous geochemical 25 reactions, collectively known as the process of serpentinization, are exothermic and are associated with the release of hydrogen gas (H 2 ), methane (CH 4 ), and small organic molecules. The biological consequences of this flux of energy and organic compounds from the Atlantis Massif were explored by International Ocean Discovery Program (IODP) Expedition 357, which used seabed drills to collect continuous 30 sequences of shallow (<16 meters below seafloor) marine serpentinites and mafic assemblages. Here, we report the first census of microbial diversity in samples of the drill cores, as measured by environmental 16S rRNA gene amplicon sequencing.The problem of contamination of subsurface samples was a primary concern during all stages of this project, starting from the initial study design, continuing to the 35 collection of samples from the seafloor, handling the samples shipboard and in the lab, preparing the samples for DNA extraction, and analyzing the DNA sequence data. To distinguish endemic microbial taxa of serpentinite subsurface rocks from seawater residents and other potential contaminants, the distributions of individual 16S rRNA gene sequences among all samples were evaluated, taking into 40 consideration both presence/absence as well as relative abundances. Our results highlight a few candidate residents of the shallow serpentinite subsurface, including uncultured representatives of the Thermoplasmata, Acidobacteria, Acidimicrobiia, and Chloroflexi. 3 45 Importance International Ocean Discovery Program Expedition 357: "Serpentinization and Life" utilized seabed drills for the first time to collect rocks from the oceanic crust. The recovered rock cores represent the shallow serpentinite subsurface of the Atlantis Massif, where reactions between uplifted mantle rocks and water, collectively known 50 as serpentinization, produce environmental conditions that can stimulate biological activity and are thought to be analogous to environments that were prevalent on the early Earth and perhaps other planets. The methodology and results of this project have implications for life detection experiments, including sample return missions, and provide the first window into the diversity of microbial communities inhabiting 55 subseafloor serpentinites.

Alkaline fluids venting from chimneys of the Lost City hydrothermal field flow from a potentially vast microbial habitat within the seafloor where energy and organic molecules are released by chemical reactions within rocks uplifted from Earth's mantle. In this study, we investigated hydrothermal fluids venting from Lost City chimneys as windows into subseafloor environments where the products of geochemical reactions, such as hydrogen (H2), formate, and methane, may be the only available sources of energy for biological activity. Our deep sequencing of metagenomes and metatranscriptomes from these hydrothermal fluids revealed a few key species of archaea and bacteria that are likely to play critical roles in the subseafloor microbial ecosystem. We identified a population of Thermodesulfovibrionales (belonging to phylum Nitrospirae) as a prevalent sulfate-reducing bacterium that may be responsible for much of the consumption of H2 and sulfate in Lost City fluids. Metagenome-assembled genomes (MAGs) classified as Methanosarcinaceae and Candidatus Bipolaricaulota were also recovered from venting fluids and represent potential methanogenic and acetogenic members of the subseafloor ecosystem. These genomes share novel hydrogenases and formate dehydrogenase-like sequences that may be unique to hydrothermal and subsurface alkaline environments where hydrogen and formate are much more abundant than carbon dioxide. The results of this study include multiple examples of metabolic strategies that appear to be advantageous in hydrothermal and subsurface environments where energy and carbon are provided by geochemical reactions.

Strýtan Hydrothermal Field (SHF) is a submarine system located in Eyjafördur in northern Iceland composed of two main vents: Big Strýtan and Arnarnesstrýtan. The vents are shallow, ranging from 16 to 70 m water depth, and vent high pH (up to 10.2), moderate temperature (Tmax ∼70°C), anoxic, fresh fluids elevated in dissolved silica, with slightly elevated concentrations of hydrogen and methane. In contrast to other alkaline hydrothermal vents, SHF is unique because it is hosted in basalt and therefore the high pH is not created by serpentinization. While previous studies have assessed the geology and geochemistry of this site, the microbial diversity of SHF has not been explored in detail. Here we present a microbial diversity survey of the actively venting fluids and chimneys from Big Strýtan and Arnarnesstrýtan, using 16S rRNA gene amplicon sequencing. Community members from the vent fluids are mostly aerobic heterotrophic bacteria; however, within the chimneys oxic, low oxygen, and anoxic habitats could be distinguished, where taxa putatively capable of acetogenesis, sulfur-cycling, and hydrogen metabolism were observed. Very few archaea were observed in the samples. The inhabitants of SHF are more similar to terrestrial hot spring samples than other marine sites. It has been hypothesized that life on Earth (and elsewhere in the solar system) could have originated in an alkaline hydrothermal system, however all other studied alkaline submarine hydrothermal systems to date are fueled by serpentinization. SHF adds to our understandings of hydrothermal vents in relationship to microbial diversity, evolution, and possibly the origin of life.

Abstract. International Ocean Discovery Program (IODP) Expedition 357: “Serpentinization and Life” drilled shallow cores into the Atlantis Massif near the Mid-Atlantic Ridge in October 2015 using seabed drills. Serpentinization and other geochemical processes occurring within the Atlantis Massif release hydrogen, methane, and other chemicals that can potentially fuel microorganisms through chemosynthesis. The subseafloor rock cores collected during IODP Exp. 357 are the first of their kind, meaning the analysis and interpretation of these samples required new methodologies, including a specialized approach for distinguishing endemic subsurface inhabitants from potential contaminants from various sources. Background samples of various potential contamination sources were collected during sampling: 109 samples of seawater collected before, during, and after drilling; 20 samples of greases and oils associated with the drilling equipment; and samples of the laboratory's ambient air. Despite the widespread usage of drilling lubricants and the importance of controlling contamination in drill-core samples for microbiological analyses, no studies to date have looked at DNA in drilling greases and oils. In this study, drilling lubricants were analyzed as possible sources of microbial contamination of subseafloor rock core samples by environmental sequencing of 16S rRNA genes. We find that microbial signatures from drilling lubricants are only found in low abundance in seafloor samples (at most a few percent of total sequence counts), with laboratory contaminants being a greater source of contamination.