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Methane is a key component in the global carbon cycle, with a wide range of anthropogenic and natural sources. Although isotopic compositions of methane have traditionally aided source identification, the abundance of its multiply substituted \"clumped\" isotopologues (for example, 13CH3D) has recently emerged as a proxy for determining methane-formation temperatures. However, the effect of biological processes on methane's clumped isotopologue signature is poorly constrained. We show that methanogenesis proceeding at relatively high rates in cattle, surface environments, and laboratory cultures exerts kinetic control on 13CH3D abundances and results in anomalously elevated formation-temperature estimates. We demonstrate quantitatively that H2 availability accounts for this effect. Clumped methane thermometry can therefore provide constraints on the generation of methane in diverse settings, including continental serpentinization sites and ancient, deep groundwaters.

Nanophase iron (oxyhydr)oxides are ubiquitous on Earth, globally distributed on Mars, and likely present on numerous other rocky solar system bodies. They are often structurally and, therefore, spectrally distinct from iron (oxyhydr)oxide bulk phases. Because their spectra vary with grain size, they can be difficult to identify or distinguish unless multiple analysis techniques are used in tandem. Yet, most literature reports fail to use multiple techniques or adequately parameterize sample morphology, making it difficult to understand how morphology affects spectral characteristics across techniques. Here, we present transmission electron microscopy, Raman, visible and near-infrared, and mid-infrared attenuated total reflectance data on synthetic, nanophase akaganéite, lepidocrocite, goethite, hematite, ferrihydrite, magnetite, and maghemite. Feature positions are tabulated and compared to those for bulk (oxyhydr)oxides and other nanophase iron (oxyhydr)oxides from the literature. The utility and limitations of each technique in analyzing nanophase iron (oxyhydr)oxides are discussed. Raman, mid-infrared, and visible near-infrared spectra show broadening, loss of some spectral features, and shifted positions compared to bulk phases. Raman and mid-infrared spectroscopies are useful in identifying and distinguishing akaganéite, lepidocrocite, goethite, and hematite, though ferrihydrite, magnetite, and maghemite have overlapped band positions. Visible near-infrared spectroscopy can identify and distinguish among ferrihydrite, magnetite, and maghemite in pure spectra, though akaganéite, lepidocrocite, and goethite can have overlapping bands. It is clear from this work that further understanding of variable spectral features in nanophase iron (oxyhydr)oxides must await additional studies to robustly assess effects of morphology. This study establishes a template for future work.

Microbial productivity at hydrothermal vents is among the highest found anywhere in the deep ocean, but constraints on microbial growth and metabolism at vents are lacking. We used a combination of cultivation, molecular, and geochemical tools to verify pure culture H ₂ threshold measurements for hyperthermophilic methanogenesis in low-temperature hydrothermal fluids from Axial Volcano and Endeavour Segment in the northeastern Pacific Ocean. Two Methanocaldococcus strains from Axial and Methanocaldococcus jannaschii showed similar Monod growth kinetics when grown in a bioreactor at varying H ₂ concentrations. Their H ₂ half-saturation value was 66 μM, and growth ceased below 17–23 μM H ₂, 10-fold lower than previously predicted. By comparison, measured H ₂ and CH ₄ concentrations in fluids suggest that there was generally sufficient H ₂ for Methanocaldococcus growth at Axial but not at Endeavour. Fluids from one vent at Axial (Marker 113) had anomalously high CH ₄ concentrations and contained various thermal classes of methanogens based on cultivation and mcrA / mrtA analyses. At Endeavour, methanogens were largely undetectable in fluid samples based on cultivation and molecular screens, although abundances of hyperthermophilic heterotrophs were relatively high. Where present, Methanocaldococcus genes were the predominant mcrA / mrtA sequences recovered and comprised ∼0.2–6% of the total archaeal community. Field and coculture data suggest that H ₂ limitation may be partly ameliorated by H ₂ syntrophy with hyperthermophilic heterotrophs. These data support our estimated H ₂ threshold for hyperthermophilic methanogenesis at vents and highlight the need for coupled laboratory and field measurements to constrain microbial distribution and biogeochemical impacts in the deep sea.

Iron–sulfur clusters are ubiquitous in biology and function in electron transfer and catalysis. They are assembled from iron and cysteine sulfur on protein scaffolds. Iron is typically stored as iron oxyhydroxide, ferrihydrite, encapsulated in 12 nm shells of ferritin, which buffers cellular iron availability. Here we have characterized IssA, a protein that stores iron and sulfur as thioferrate, an inorganic anionic polymer previously unknown in biology. IssA forms nanoparticles reaching 300 nm in diameter and is the largest natural metalloprotein complex known. It is a member of a widely distributed protein family that includes nitrogenase maturation factors, NifB and NifX. IssA nanoparticles are visible by electron microscopy as electron-dense bodies in the cytoplasm. Purified nanoparticles appear to be generated from 20 nm units containing ∼6,400 Fe atoms and ∼170 IssA monomers. In support of roles in both iron–sulfur storage and cluster biosynthesis, IssA reconstitutes the [4Fe-4S] cluster in ferredoxin in vitro . The biosynthesis of iron-sulfur clusters in anaerobic organisms has not been extensively investigated. Here, the authors identify and characterize a multi-subunit protein that stores iron and sulfur in thioferrate for the assembly of the clusters in Pyrococcus furiosus .

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 CH 4 production kinetics based on chemostat H 2 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 H 2 , CH 4 , 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 10 11 methanogenic cells occupying 1.8–18 m 3 of ocean crust per m 2 of vent seafloor area were needed to produce the observed CH 4 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.

A deep-sea thermophilic bacterium, strain Ax17T, was isolated from 25 °C hydrothermal fluid at Axial Seamount. It was obligately anaerobic and autotrophic, oxidized molecular hydrogen and formate, and reduced synthetic nanophase Fe(III) (oxyhydr)oxide minerals, sulfate, sulfite, thiosulfate, and elemental sulfur for growth. It produced up to 20 mM Fe2+ when grown on ferrihydrite but < 5 mM Fe2+ when grown on akaganéite, lepidocrocite, hematite, and goethite. It was a straight to curved rod that grew at temperatures ranging from 35 to 70 °C (optimum 65 °C) and a minimum doubling time of 7.1 h, in the presence of 1.5–6% NaCl (optimum 3%) and pH 5–9 (optimum 8.0). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was 90–92% identical to other genera of the family Desulfonauticaceae in the phylum Pseudomonadota. The genome of Ax17T was sequenced, which yielded 2,585,834 bp and contained 2407 protein-coding sequences. Based on overall genome relatedness index analyses and its unique phenotypic characteristics, strain Ax17T is suggested to represent a novel genus and species, for which the name Desulfovulcanus ferrireducens is proposed. The type strain is Ax17T (= DSM 111878T = ATCC TSD-233T).

The deduced amino acid sequence from a gene of the hyperthermophilic archaeon Pyrococcus sp. ST04 (Py04_0872) contained a conserved glycoside hydrolase family 57 (GH57) motif, but showed <13 % sequence identity with other known Pyrococcus GH57 enzymes, such as 4-α-glucanotransferase (EC 2.4.1.25), amylopullulanase (EC 3.2.1.41), and branching enzyme (EC 2.4.1.18). This gene was cloned and expressed in Escherichia coli, and the recombinant product (P yrococcus sp. ST04 maltose-forming α-amylase, PSMA) was a novel 70-kDa maltose-forming α-amylase. PSMA only recognized maltose (G2) units with α-1,4 and α-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzed pullulan very poorly. G2 was the primary end product of hydrolysis. Branched cyclodextrin (CD) was only hydrolyzed along its branched maltooligosaccharides. 6-O-glucosyl-β-cyclodextrin (G1-β-CD) and β-cyclodextrin (β-CD) were resistant to PSMA suggesting that PSMA is an exo-type glucan hydrolase with α-1,4- and α-1,6-glucan hydrolytic activities. The half-saturation value (K ₘ) for the α-1,4 linkage of maltotriose (G3) was 8.4 mM while that of the α-1,6 linkage of 6-O-maltosyl-β-cyclodextrin (G2-β-CD) was 0.3 mM. The k cₐₜ values were 381.0 min⁻¹ for G3 and 1,545.0 min⁻¹ for G2-β-CD. The enzyme was inhibited competitively by the reaction product G2, and the K ᵢ constant was 0.7 mM. PSMA bridges the gap between amylases that hydrolyze larger maltodextrins and α-glucosidase that feeds G2 into glycolysis by hydrolyzing smaller glucans into G2 units.

Endeavour Segment of the Juan de Fuca Ridge is one of three Integrated Study Sites for the Ridge 2000 Program. It is a remarkable, dynamic environment hosting five major hydrothermal fields, numerous smaller fields, and myriad diffuse-flow sites; magma chambers underlie all fields. Over 800 individual extinct and active chimneys have been documented within the central ~ 15 km portion of the ridge, with some edifices reaching 50 m across and up to 45 m tall. Fluid flow is focused along faults within the rift zone, and seismically active faults along the western axial valley wall have been used by both magmas and upwelling hydrothermal fluids. There is significant chemical heterogeneity in basalt compositions within the axial rift valley, with the greatest diversity occurring near the base of the western axial valley wall where normal, transitional, and enriched type mid-ocean ridge basalts occur within tens of meters of each other. Endeavour is the only site where seismic intensity has been linked directly to heat flux at the individual vent field scale. Installation of the world's first high-power and high-bandwidth cabled observatory at Endeavour via NEPTUNE Canada ensures that new discoveries along the Juan de Fuca Ridge will continue into the future.

Hydrothermal vents are among the most biologically active regions of the deep ocean. However, our understanding of the limits of life in this extreme environment, the extent of biogeochemical transformation that occurs in the crust and overlying ocean, and the impact of vent life on regional and global ocean chemistry is in its infancy. Recently, scientific studies have expanded our view of how vent microbes gain metabolic energy at vents through their use of dissolved chemicals and minerals contained in ocean basalts, seafloor sulfide deposits, and hydrothermal plumes and, in turn, how they catalyze chemical and mineral transformations. The scale of vent environments and the difficulties inherent in the study of life above, on, and below the deep seafloor have led to the development of geochemical and bioenergetic models. These models predict habitability and biological activity based on the chemical composition of hydrothermal fluids, seawater, and the surrounding rock, balanced by the physiological energy demand of cells. This modeling, coupled with field sampling for ground truth and discovery, has led to a better understanding of how hydrothermal vents affect the ocean and global geochemical cycles, and how they influence our views of life on the early Earth and the search for life beyond our own planet.

Abstract Eight new strains of deep-sea hyperthermophilic sulfur reducers were isolated from hydrothermal vent fields at 9°50′N East Pacific Rise (EPR) and at the Cleft and CoAxial segments along the Juan de Fuca Ridge (JdFR). 16S rRNA gene sequence analysis showed that each strain belongs to the genus Thermococcus. Restriction fragment length polymorphism patterns of the 16S/23S rRNA intergenic spacer region revealed that these isolates fell into three groups: those from the EPR, those from fluid and rock sources on the JdFR, and those isolated from Paralvinella spp. polychaete vent worms from the JdFR. The optimum-temperature specific growth rates and the temperature ranges for growth were significantly higher and broader for those strains isolated from worms relative to those isolated from low-temperature diffuse hydrothermal fluids. Furthermore, the worm-derived isolates generally produced a larger array of proteases and amylases based on zymogram analyses. The zymogram patterns also changed with growth temperature suggesting that these organisms alter their lytic protein suites in response to changes in temperature. This study suggests that there is significant phenotypic diversity in Thermococcus that is not apparent from their highly conserved 16S rRNA nucleotide sequences.