David A. Butterfield

At deep-sea hydrothermal vents, magmatically driven rock-water reactions in the crust generate gases and other reduced compounds that subseafloor microorganisms use for chemolithoautotrophy. In this study, microbial autotrophs from three diffuse flow hydrothermal vents at Axial Seamount in 2013 and 2014 were isotopically labeled using RNA stable isotope probing, targeting subseafloor autotrophic mesophiles (30°C), thermophiles (55°C), and hyperthermophiles (80°C). We constructed taxonomic and functional profiles of active chemolithoautotrophs, examined population distributions across sites, and linked primary producers to their specific metabolic strategies within the subseafloor community. Dominant autotrophs exhibited hydrogen-dependent dissimilatory metabolisms, such as sulfur and nitrate reduction and methanogenesis, as well as microaerophilic sulfide oxidation even at 80°C, consistent with fluid chemistries at each site. While hydrogenotrophic methanogenic archaea ( Methanothermococcus ) were restricted in their distribution and activity, hydrogenotrophic sulfur and nitrate reducers from the Aquificota ( Thermovibrio ) and Campylobacterota (Nautiliaceae, Hydrogenimonas , and Desulfurobacteriaceae) were consistently active and present at all sites and years at both the population and community levels. Hydrogenase transcripts were significantly differentially expressed, and diverse hydrogenases were found in metagenome-assembled genomes of Aquificota members, highlighting the importance and versatility of their hydrogen utilization strategies, which likely contribute to their cosmopolitan distribution across geochemically disparate subseafloor sites. Together, this study provides new insights into the functional dynamics and distribution of key subseafloor autotrophic microbial communities in young oceanic crust at deep-sea hydrothermal vents. IMPORTANCE: Deep-sea hydrothermal vents are hotspots for life in the dark ocean, where rich animal ecosystems are supported by microbial primary producers utilizing the abundant chemical energy supplied by high-temperature water-rock reactions. Despite increasing knowledge about the geochemistry and microbiology of deep-sea hydrothermal vents, there is still a gap in our understanding of the key microbial players who fix much of the carbon at these sites, especially in the productive subseafloor. In this study, stable isotope probing was used to label active microbial autotrophs in diffuse flow venting fluids from three sites over 2 years and was combined with metatranscriptomic sequencing to identify their specific metabolic strategies. This research highlights the microbial community composition, function, gene regulation, and population dynamics that enable hydrothermal ecosystems to persist.

At deep-sea hydrothermal vents, magmatically driven rock-water reactions in the crust generate gases and other reduced compounds that subseafloor microorganisms use for chemolithoautotrophy. In this study, microbial autotrophs from three diffuse flow hydrothermal vents at Axial Seamount in 2013 and 2014 were isotopically labeled using RNA Stable Isotope Probing (RNA-SIP), targeting subseafloor autotrophic mesophiles (30°C), thermophiles (55°C), and hyperthermophiles (80°C). We constructed taxonomic and functional profiles of active chemolithoautotrophs, examined population distributions across sites, and linked primary producers to their specific metabolic strategies within the subseafloor community. Dominant autotrophs exhibited hydrogen-dependent dissimilatory metabolisms such as sulfur and nitrate reduction and methanogenesis, as well as microaerophilic sulfide oxidation even at 80°C, consistent with fluid chemistries at each site. While hydrogenotrophic methanogenic archaea such as Methanothermococcus were restricted in their distribution and activity, hydrogenotrophic sulfur and nitrate reducers from the Aquificota ( Thermovibrio ), Campylobacterota (Nautiliaceae, Hydrogenimonas , and Desulfurobacteriaceae) were consistently active and present at all sites and years at both the population and community levels. Hydrogenase transcripts were significantly differentially expressed, and diverse hydrogenases were found in metagenome-assembled genomes of Aquificota members, highlighting the importance and versatility of their hydrogen utilization strategies which likely contribute to their cosmopolitan distribution across geochemically disparate subseafloor sites. Together, this study provides new insights into the functional dynamics and distribution of key subseafloor autotrophic microbial communities in young oceanic crust at deep-sea hydrothermal vents. IMPORTANCE: Deep-sea hydrothermal vents are hotspots for life in the dark ocean, where rich animal ecosystems are supported by microbial primary producers utilizing the abundant chemical energy supplied by high-temperature water-rock reactions. Despite increasing knowledge about the geochemistry and microbiology of deep-sea hydrothermal vents, there is still a gap in our understanding of the key microbial players who fix much of the carbon at these sites, especially in the productive subseafloor. In this study, stable isotope probing was used to label active microbial autotrophs in diffuse flow venting fluids from three sites over two years and was combined with metatranscriptomic sequencing to identify their specific metabolic strategies. This research highlights the microbial community composition, function, gene regulation, and population dynamics that enable hydrothermal ecosystems to persist.

Single‐celled microbial eukaryotes inhabit deep‐sea hydrothermal vent environments and play critical ecological roles in the vent‐associated microbial food web. 18S rRNA amplicon sequencing of diffuse venting fluids from four geographically‐ and geochemically‐distinct hydrothermal vent fields was applied to investigate community diversity patterns among protistan assemblages. The four vent fields include Axial Seamount at the Juan de Fuca Ridge, Sea Cliff and Apollo at the Gorda Ridge, all in the NE Pacific Ocean, and Piccard and Von Damm at the Mid‐Cayman Rise in the Caribbean Sea. We describe species diversity patterns with respect to hydrothermal vent field and sample type, identify putative vent endemic microbial eukaryotes, and test how vent fluid geochemistry may influence microbial community diversity. At a semi‐global scale, microbial eukaryotic communities at deep‐sea vents were composed of similar proportions of dinoflagellates, ciliates, Rhizaria, and stramenopiles. Individual vent fields supported distinct and highly diverse assemblages of protists that included potentially endemic or novel vent‐associated strains. These findings represent a census of deep‐sea hydrothermal vent protistan communities. Protistan diversity, which is shaped by the hydrothermal vent environment at a local scale, ultimately influences the vent‐associated microbial food web and the broader deep‐sea carbon cycle.

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 (H ₂ ), 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 H ₂ 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. IMPORTANCE: 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 hydrogen and creates unusual environmental conditions where simple organic carbon molecules are more stable than dissolved inorganic carbon. This study provides an initial glimpse into the kinds of microbes that live deep within the seafloor where serpentinization takes place, by sampling hydrothermal fluids exiting from the Lost City chimneys. The metabolic strategies that these microbes appear to be using are also shared by microbes that inhabit other sites of serpentinization, including continental subsurface environments and natural springs. Therefore, the results of this study contribute to a broader, interdisciplinary effort to understand the general principles and mechanisms by which serpentinization-associated processes can support life on Earth and perhaps other worlds.

The impact of submarine hydrothermal systems on organic carbon in the ocean—one of the largest fixed carbon reservoirs on Earth—could be profound. Yet, different vent sites show diverse fluid chemical compositions and the subsequent biological responses. Observations from various vent sites are to evaluate hydrothermal systems' impact on the ocean carbon cycle. A response cruise in May 2009 to an on‐going submarine eruption at West Mata Volcano, northeast Lau Basin, provided an opportunity to quantify the organic matter production in a back‐arc spreading hydrothermal system. Hydrothermal vent fluids contained elevated dissolved organic carbon, particulate organic carbon (POC), and particulate nitrogen (PN) relative to background seawater. The δ¹³C‐POC values for suspended particles in the diffuse vent fluids (−15.5‰ and −12.3‰) are distinct from those in background seawater (−23 ± 1‰), indicative of unique carbon synthesis pathways of the vent microbes from the seawater counterparts. The first dissolved organic nitrogen concentrations reported for diffuse vents were similar to or higher than those for background seawater. Enhanced nitrogen fixation and denitrification removed 37%–89% of the total dissolved nitrogen in the recharging background seawater in the hydrothermal vent flow paths. The hydrothermal plume samples were enriched in POC and PN, indicating enhanced biological production. The total “dark” organic carbon production within the plume matches the thermodynamic prediction based on available reducing chemical substances supplied to the plume. This research combines the measured organic carbon contents with thermodynamic modeled results and demonstrates the importance of hydrothermal activities on the water column carbon production in the deep ocean.

Current methods for biochemical and biogeochemical analysis of the deep-sea hydrothermal vent ecosystems rely on water sample recovery, or in situ analysis using underwater instruments with limited range of analyte detection and limited sensitivity. Even in cases where large quantities of sample are recovered, labile dissolved organic compounds may not be detected due to time delays between sampling and preservation. Here, we present a novel approach for in situ extraction of organic compounds from hydrothermal vent fluids through a unique solid phase microextraction (SPME) sampler. These samplers were deployed to sample effluent of vents on sulphide chimneys, located on Axial Seamount in the North-East Pacific, in the Urashima field on the southern Mariana back-arc, and at the Hafa Adai site in the central Mariana back-arc. Among the compounds that were extracted, a wide range of unique organic compounds, including labile dissolved organic sulfur compounds, were detected through high-resolution LC-MS/MS, among which were biomarkers of anammox bacteria, fungi, and lower animals. This report is the first to show that SPME can contribute to a broader understanding of deep sea ecology and biogeochemical cycles in hydrothermal vent ecosystems.

There are many unknowns regarding the distribution, activity, community composition, and metabolic repertoire of microbial communities in the subseafloor of deep-sea hydrothermal vents. Here we provide the first characterization of subseafloor microbial communities from venting fluids along the central Mariana back-arc basin (15.5–18°N), where the slow-spreading rate, depth, and variable geochemistry along the back-arc distinguish it from other spreading centers. Results indicated that diverse Epsilonbacteraeota were abundant across all sites, with a population of high temperature Aquificae restricted to the northern segment. This suggests that differences in subseafloor populations along the back-arc are associated with local geologic setting and resultant geochemistry. Metatranscriptomics coupled to stable isotope probing revealed bacterial carbon fixation linked to hydrogen oxidation, denitrification, and sulfide or thiosulfate oxidation at all sites, regardless of community composition. NanoSIMS (nanoscale secondary ion mass spectrometry) incubations at 80 °C show only a small portion of the microbial community took up bicarbonate, but those autotrophs had the highest overall rates of activity detected across all experiments. By comparison, acetate was more universally utilized to sustain growth, but within a smaller range of activity. Together, results indicate that microbial communities in venting fluids from the Mariana back-arc contain active subseafloor communities reflective of their local conditions with metabolisms commonly shared across geologically disparate spreading centers throughout the ocean.

The size and biogeochemical impact of the subseafloor biosphere in oceanic crust remain largely unknown due to sampling limitations. We used reactive transport modeling to estimate the size of the subseafloor methanogen population, volume of crust occupied, fluid residence time, and nature of the subsurface mixing zone for two low-temperature hydrothermal vents at Axial Seamount. Monod CH₄ production kinetics based on chemostat H₂ availability and batch-culture Arrhenius growth kinetics for the hyperthermophile Methanocaldococcus jannaschii and thermophile Methanothermococcus thermolithotrophicus were used to develop and parameterize a reactive transport model, which was constrained by field measurements of H₂, CH₄, and metagenome methanogen concentration estimates in 20–40 °C hydrothermal fluids. Model results showed that hyperthermophilic methanogens dominate in systems where a narrow flow path geometry is maintained, while thermophilic methanogens dominate in systems where the flow geometry expands. At Axial Seamount, the residence time of fluid below the surface was 29–33 h. Only 10¹¹ methanogenic cells occupying 1.8–18 m³ of ocean crust per m² of vent seafloor area were needed to produce the observed CH₄ anomalies. We show that variations in local geology at diffuse vents can create fluid flow paths that are stable over space and time, harboring persistent and distinct microbial communities.

Mid‐ocean ridge eruptions, initiating or revitalizing hydrothermal discharge and disrupting seafloor ecosystems, occur regularly as a consequence of plate spreading. Evaluating their impact on long‐term hydrothermal discharge requires information on the scale and duration of any posteruption enhancement. Here we describe a unique hydrothermal plume time series of annual (or more frequent) observations at Axial Seamount vent fields from 1985 through 2017, missing only 7 years. Axial, a hot spot volcano astride the Juan de Fuca Ridge, experienced eruptions in 1998, 2011, and 2015. In 1998 and 2011 lava flooded the SE caldera and south rift zone, but in 2015 most lava was extruded in a series of flows extending ~20 km down the north rift zone. Response cruises occurred within 18 days (1998) to about 4 months, followed by regular posteruption observations. All 30 cruises measured plume rise height (a proxy for heat flux) and turbidity (indicative of chemical changes in vent discharge) at several vent sites, yielding an integrated view of vent field activity. Venting in the SE caldera area persisted throughout the time series, consistent with the imaged location of the shallowest portion of the melt‐rich magma reservoir. Eruptions produced substantial and diagnostic increases in plume rise and turbidity, and posteruption enhancements lasted 2–5 years, totaling ~10 years over the course of the time series. Estimates of the relative heat flux indicate a sixfold increase during eruption‐enhanced periods, implying that generalizations about mid‐ocean ridge hydrothermal fluxes may be underestimates if based on non–eruption‐enhanced hydrothermal activity alone.