Microbe-Mineral Interaction and Novel Proteins for Iron Oxide Mineral Reduction in the Hyperthermophilic Crenarchaeon \u3cem\u3ePyrodictium delaneyi\u3c/em\u3e

Dissimilatory iron reduction by hyperthermophilic archaea occurs in many geothermal environments and generally relies on microbe-mineral interactions that transform various iron oxide minerals. In this study, the physiology of dissimilatory iron and nitrate reduction was examined in the hyperthermophilic crenarchaeon type strain Pyrodictium delaneyi Su06. Iron barrier experiments showed that P. delaneyi required direct contact with the Fe(III) oxide mineral ferrihydrite for reduction. The separate addition of an exogenous electron shuttle (anthraquinone-2,6-disulfonate), a metal chelator (nitrilotriacetic acid), and 75% spent cell-free supernatant did not stimulate growth with or without the barrier. Protein electrophoresis showed that the c-type cytochrome and general protein compositions of P. delaneyi changed when grown on ferrihydrite relative to nitrate. Differential proteomic analyses using tandem mass tagged protein fragments and mass spectrometry detected 660 proteins and differential production of 127 proteins. Among these, two putative membrane-bound molybdopterin-dependent oxidoreductase complexes increased in relative abundance 60- to 3,000-fold and 50- to 100-fold in cells grown on iron oxide. A putative 8-heme c-type cytochrome was 60-fold more abundant in iron-grown cells and was unique to the Pyrodictiaceae. There was also a \u3e14,700-fold increase in a membrane transport protein in iron-grown cells. For flagellin proteins and a putative nitrate reductase, there were no changes in abundance, but a membrane nitric oxide reductase was more abundant on nitrate. These data help to elucidate the mechanisms by which hyperthermophilic crenarchaea generate energy and transfer electrons across the membrane to iron oxide minerals

Draft Genome Sequence of \u3cem\u3eDesulfurobacterium thermolithotrophum\u3c/em\u3e Strain HR11, a Novel Thermophilic Autotrophic Subspecies from a Deep-Sea Hydrothermal Vent

Desulfurobacterium sp. strain HR11 was isolated from a hydrothermal vent on the Juan de Fuca Ridge. We present the 1.55-Mb genome sequence of HR11, which contains 1,624 putative protein-coding sequences. Overall genome relatedness index analyses indicate that HR11 is a novel subspecies of D. thermolithotrophum

Growth Kinetics, Carbon Isotope Fractionation, and Gene Expression in the Hyperthermophile Methanocaldococcus jannaschii during Hydrogen-Limited Growth and Interspecies Hydrogen Transfer

Hyperthermophilic methanogens are often H2 limited in hot subseafloor environments, and their survival may be due in part to physiological adaptations to low H2 conditions and interspecies H2 transfer. The hyperthermophilic methanogen Methanocaldococcus jannaschii was grown in monoculture at high (80 to 83 μM) and low (15 to 27 μM) aqueous H2 concentrations and in coculture with the hyperthermophilic H2 producer Thermococcus paralvinellae. The purpose was to measure changes in growth and CH4 production kinetics, CH4 fractionation, and gene expression in M. jannaschii with changes in H2 flux. Growth and cell-specific CH4 production rates of M. jannaschii decreased with decreasing H2 availability and decreased further in coculture. However, cell yield (cells produced per mole of CH4 produced) increased 6-fold when M. jannaschii was grown in coculture rather than monoculture. Relative to high H2 concentrations, isotopic fractionation of CO2 to CH4(εCO2-CH4) was 16‰ larger for cultures grown at low H2 concentrations and 45‰ and 56‰ larger for M. jannaschii growth in coculture on maltose and formate, respectively. Gene expression analyses showed H2-dependent methylene-tetrahydromethanopterin (H4MPT) dehydrogenase expression decreased and coenzyme F420-dependent methylene-H4MPT dehydrogenase expression increased with decreasing H2 availability and in coculture growth. In coculture, gene expression decreased for membrane-bound ATP synthase and hydrogenase. The results suggest that H2 availability significantly affects the CH4 and biomass production and CH4 fractionation by hyperthermophilic methanogens in their native habitats

Introduction to the special issue : From RIDGE to Ridge 2000

Author Posting. © The Oceanography Society, 2012. This article is posted here by permission of The Oceanography Society for personal use, not for redistribution. The definitive version was published in Oceanography 25, no. 1 (2012): 12–17, doi:10.5670/oceanog.2012.01.Articles in this special issue of Oceanography represent a compendium of research that spans the disciplinary and thematic breadth of the National Science Foundation's Ridge 2000 Program, as well as its geographic focal points. The mid-ocean ridge (MOR) crest is where much of Earth's volcanism is focused and where most submarine volcanic activity occurs. If we could look down from space at our planet with the ocean drained, the MOR's topography and shape, along with its intervening fracture zones, would resemble the seams on a baseball, with the ocean basins dominating our planetary panorama. The volcanic seafloor is hidden beneath the green-blue waters of the world's ocean, yet therein lie fundamental clues to how our planet works and has evolved over billions of years, something that was not clearly understood 65 years ago—witness the following quote from H.H. Hess (1962) in his essay on "geopoetry" and commentary on J.H.F. Umbgrove's (1947) comprehensive summary of Earth and ocean history: The birth of the oceans is a matter of conjecture, the subsequent history is obscure, and the present structure is just beginning to be understood. Fascinating speculation on these subjects has been plentiful, but not much of it predating the last decade [the 1950s] holds water.This special issue was funded by a supplement to the Ridge 2000 Office grant at the Woods Hole Oceanographic Institution (NSF-OCE-0838923)

Biogeochemical processes at hydrothermal vents : microbes and minerals, bioenergetics, and carbon fluxes

Author Posting. © The Oceanography Society, 2012. This article is posted here by permission of The Oceanography Society for personal use, not for redistribution. The definitive version was published in Oceanography 25, no. 1 (2012): 196–208, doi:10.5670/oceanog.2012.18.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.Research for this paper was supported by the National Science Foundation (NSF) Division of Ocean Sciences grants 0732611 for JFH, 0926805 and 1038055 for JAB, and 1038055 for BMT; and by the University of Missouri Research Board for KLR

Hydrogen Production and Enzyme Activities in the Hyperthermophile Thermococcus paralvinellae Grown on Maltose, Tryptone, and Agricultural Waste

Thermococcus may be an important alternative source of H2 in the hot subseafloor in otherwise low H2environments such as some hydrothermal vents and oil reservoirs. It may also be useful in industry for rapid agricultural waste treatment and concomitant H2 production. Thermococcus paralvinellae grown at 82°C without sulfur produced up to 5 mmol of H2 L−1 at rates of 5–36 fmol H2 cell−1 h−1 on 0.5% (wt vol−1) maltose, 0.5% (wt vol−1) tryptone, and 0.5% maltose + 0.05% tryptone media. Two potentially inhibiting conditions, the presence of 10 mM acetate and low pH (pH 5) in maltose-only medium, did not significantly affect growth or H2production. Growth rates, H2 production rates, and cell yields based on H2 production were the same as those for Pyrococcus furiosus grown at 95°C on the same media for comparison. Acetate, butyrate, succinate, isovalerate, and formate were also detected as end products. After 100 h, T. paralvinellae produced up to 5 mmol of H2 L−1 of medium when grown on up to 70% (vol vol−1) waste milk from cows undergoing treatment for mastitis with the bacterial antibiotic Ceftiofur and from untreated cows. The amount of H2 produced by T. paralvinellae increased with increasing waste concentrations, but decreased in P. furiosus cultures supplemented with waste milk above 1% concentration. All mesophilic bacteria from the waste milk that grew on Luria Bertani, Sheep\u27s Blood (selective for Staphylococcus, the typical cause of mastitis), and MacConkey (selective for Gram-negative enteric bacteria) agar plates were killed by heat during incubation at 82°C. Ceftiofur, which is heat labile, was below the detection limit following incubation at 82°C. T. paralvinellae also produced up to 6 mmol of H2 L−1 of medium when grown on 0.1–10% (wt vol−1) spent brewery grain while P. furiosus produced \u3c 1 mmol of H2 L−1. Twelve of 13 enzyme activities in T. paralvinellae showed significant (p \u3c 0.05) differences across six different growth conditions; however, methyl viologen-dependent membrane hydrogenase activity remained constant across all media types. The results demonstrate the potential of at least some Thermococcus species to produce H2 if protein and α-glucosides are present as substrates

Fe(III) (oxyhydr)oxide reduction by the thermophilic iron-reducing bacterium Desulfovulcanus ferrireducens

Some thermophilic bacteria from deep-sea hydrothermal vents grow by dissimilatory iron reduction, but our understanding of their biogenic mineral transformations is nascent. Mineral transformations catalyzed by the thermophilic iron-reducing bacterium Desulfovulcanus ferrireducens during growth at 55°C were examined using synthetic nanophase ferrihydrite, akaganeite, and lepidocrocite separately as terminal electron acceptors. Spectral analyses using visible-near infrared (VNIR), Fourier-transform infrared attenuated total reflectance (FTIR-ATR), and Mössbauer spectroscopies were complemented with x-ray diffraction (XRD) and transmission electron microscopy (TEM) using selected area electron diffraction (SAED) and energy dispersive X-ray (EDX) analyses. The most extensive biogenic mineral transformation occurred with ferrihydrite, which produced a magnetic, visibly dark mineral with spectral features matching cation-deficient magnetite. Desulfovulcanus ferrireducens also grew on akaganeite and lepidocrocite and produced non-magnetic, visibly dark minerals that were poorly soluble in the oxalate solution. Bioreduced mineral products from akaganeite and lepidocrocite reduction were almost entirely absorbed in the VNIR spectroscopy in contrast to both parent minerals and the abiotic controls. However, FTIR-ATR and Mössbauer spectra and XRD analyses of both biogenic minerals were almost identical to the parent and control minerals. The TEM of these biogenic minerals showed the presence of poorly crystalline iron nanospheres (50–200 nm in diameter) of unknown mineralogy that were likely coating the larger parent minerals and were absent from the controls. The study demonstrated that thermophilic bacteria transform different types of Fe(III) (oxyhydr)oxide minerals for growth with varying mineral products. These mineral products are likely formed through dissolution-reprecipitation reactions but are not easily predictable through chemical equilibrium reactions alone

Hydrogen limitation and syntrophic growth among natural assemblages of thermophilic methanogens at deep-sea hydrothermal vents

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 7 (2016): 1240, doi:10.3389/fmicb.2016.01240.Thermophilic methanogens are common autotrophs at hydrothermal vents, but their growth constraints and dependence on H2 syntrophy in situ are poorly understood. Between 2012 and 2015, methanogens and H2-producing heterotrophs were detected by growth at 80∘C and 55∘C at most diffuse (7–40∘C) hydrothermal vent sites at Axial Seamount. Microcosm incubations of diffuse hydrothermal fluids at 80∘C and 55∘C demonstrated that growth of thermophilic and hyperthermophilic methanogens is primarily limited by H2 availability. Amendment of microcosms with NH4+ generally had no effect on CH4 production. However, annual variations in abundance and CH4 production were observed in relation to the eruption cycle of the seamount. Microcosm incubations of hydrothermal fluids at 80∘C and 55∘C supplemented with tryptone and no added H2 showed CH4 production indicating the capacity in situ for methanogenic H2 syntrophy. 16S rRNA genes were found in 80∘C microcosms from H2-producing archaea and H2-consuming methanogens, but not for any bacteria. In 55∘C microcosms, sequences were found from H2-producing bacteria and H2-consuming methanogens and sulfate-reducing bacteria. A co-culture of representative organisms showed that Thermococcus paralvinellae supported the syntrophic growth of Methanocaldococcus bathoardescens at 82∘C and Methanothermococcus sp. strain BW11 at 60∘C. The results demonstrate that modeling of subseafloor methanogenesis should focus primarily on H2 availability and temperature, and that thermophilic H2 syntrophy can support methanogenesis within natural microbial assemblages and may be an important energy source for thermophilic autotrophs in marine geothermal environments.This work was funded by the Gordon and Betty Moore Foundation grant GBMF 3297, the NASA Earth and Space Science Fellowship Program grant NNX11AP78H, the National Science Foundation grant OCE-1547004, with funding from NOAA/PMEL, contribution #4493, and JISAO under NOAA Cooperative Agreement NA15OAR4320063, contribution #2706

Experimental investigation on the controls of clumped isotopologue and hydrogen isotope ratios in microbial methane

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Geochimica et Cosmochimica Acta 237 (2018): 339-356, doi:10.1016/j.gca.2018.06.029.The abundance of methane isotopologues with two rare isotopes (e.g., 13CH3D) has been proposed as a tool to estimate the temperature at which methane is formed or thermally equilibrated. It has been shown, however, that microbial methane from surface environments and from laboratory cultures is characterized by low 13CH3D abundance, corresponding to anomalously high apparent 13CH3D equilibrium temperatures. We carried out a series of batch culture experiments to investigate the origin of the non-equilibrium signals in microbial methane by exploring a range of metabolic pathways, growth temperatures, and hydrogen isotope compositions of the media. We found that thermophilic methanogens (Methanocaldococcus jannaschii, Methanothermococcus thermolithotrophicus, and Methanocaldococcus bathoardescens) grown on H2+CO2 at temperatures between 60 and 80°C produced methane with Δ13CH3D values (defined as the deviation from stochastic abundance) of 0.5 to 2.5‰, corresponding to apparent 13CH3D equilibrium temperatures of 200 to 600°C. Mesophilic methanogens (Methanosarcina barkeri and Methanosarcina mazei) grown on H2+CO2, acetate, or methanol produced methane with consistently low Δ13CH3D values, down to -5.2‰. Closed system effects can explain part of the non-equilibrium signals for methane from thermophilic methanogens. Experiments with M. barkeri using D-spiked water or D-labeled acetate (CD3COO-) indicate that 1.6 to 1.9 out of four H atoms in methane originate from water, but Δ13CH3D values of product methane only weakly correlate with the D/H ratio of medium water. Our experimental results demonstrate that low Δ13CH3D values are not specific to the metabolic pathways of methanogenesis, suggesting that they could be produced during enzymatic reactions common in the three methanogenic pathways, such as the reduction of methyl-coenzyme M. Nonetheless C-H bonds inherited from precursor methyl groups may also carry part of non-equilibrium signals.Grants from the National Science Foundation (EAR-1250394 to S.O.), N. Braunsdorf and D. Smit of Shell PTI/EG (to S.O.), the Deep Carbon Observatory (to S.O., M.K., K.-U.H., D.S.G.), the Gottfried Wilhelm Leibniz Program of the Deutsche Forschungsgemeinschaft (HI 616-14-1 to K.- U.H.), and the Heisenberg Program (KO3651-3-1 to M.K.) of the Deutsche Forschungsgemeinschaft supported this study. D.S.G. was also supported by a National Science Foundation Graduate Research Fellowship, the Neil and Anna Rasmussen Foundation Fund, the Grayce B. Kerr Fellowship, and a Shell-MIT Energy Initiative Graduate Fellowship. D.T.W. was supported by a National Defense Science and Engineering Graduate Fellowship. L.C.S. was supported by a NASA Earth and Space Science Fellowship (grant NNX11AP78H)

Fluid geochemistry, local hydrology, and metabolic activity define methanogen community size and composition in deep-sea hydrothermal vents

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