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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.

Extremely thermophilic methanogens in the Methanococci and heterotrophs in the Thermococci are common in deep-sea hydrothermal vents. All Methanococci use H2 as an electron donor, and a few species can also use formate. Most Methanococci have a coenzyme F420-reducing formate dehydrogenase. All Thermococci reduce S0 but have hydrogenases and produce H2 in the absence of S0. Some Thermococci have formate hydrogenlyase (Fhl) that reversibly converts H2 and CO2 to formate or an NAD(P)+-reducing formate dehydrogenase (Nfd). Questions remain if Methanococci or Thermococci use or produce formate in nature, why only certain species can grow on or produce formate, and what the physiological role of formate is? Formate forms abiotically in hydrothermal fluids through chemical equilibrium with primarily H2, CO2, and CO and is strongly dependent upon H2 concentration, pH, and temperature. Formate concentrations are highest in hydrothermal fluids where H2 concentrations are also high, such as in ultramafic systems where serpentinization reactions occur. In nature, Methanococci are likely to use formate as an electron donor when H2 is limiting. Thermococci with Fhl likely convert H2 and CO2 to formate when H2 concentrations become inhibitory for growth. They are unlikely to grow on formate in nature unless formate is more abundant than H2 in the environment. Nearly all Methanococci and Thermococci have a gene for at least one formate dehydrogenase catalytic subunit, which may be used to provide free formate for de novo purine biosynthesis. However, only species with a membrane-bound formate transporter can grow on or secrete formate. Interspecies H2 transfer occurs between Thermococci and Methanococci. This and putative interspecies formate transfer may support Methanococci in low H2 environments, which in turn may prevent growth inhibition of Thermococci by its own H2. Future research directions include understanding when, where, and how formate is used and produced by these organisms in nature, and how transcription of Thermococci genes encoding formate-related enzymes are regulated.

The 4-α-glucanotransferase (4-α-GTase or amylomaltase) is an essential enzyme in maltodextrin metabolism. Generally, most bacterial 4-α-GTase is classified into glycoside hydrolase (GH) family 77. However, hyperthermophiles have unique 4-α-GTases belonging to GH family 57. These enzymes are the main amylolytic protein in hyperthermophiles, but their mode of action in maltooligosaccharide utilization is poorly understood. In the present study, we investigated the catalytic properties of 4-α-GTase from the hyperthermophile Pyrococcus sp. ST04 (PSGT) in the presence of maltooligosaccharides of various lengths. Unlike 4-α-GTases in GH family 77, GH family 57 PSGT produced maltotriose in the early stage of reaction and preferred maltose and maltotriose over glucose as the acceptor. The kinetic analysis showed that maltotriose had the lowest KM value, which increased amylose degradation activity by 18.3-fold. Structural models of PSGT based on molecular dynamic simulation revealed two aromatic amino acids interacting with the substrate at the +2 and +3 binding sites, and the mutational study demonstrated they play a critical role in maltotriose binding. These results clarify the mode of action in carbohydrate utilization and explain acceptor binding mechanism of GH57 family 4-α-GTases in hyperthermophilic archaea.

Fe(III) (oxyhydr)oxides are electron acceptors for some hyperthermophilic archaea in mildly reducing geothermal environments. However, the kinds of iron oxides that can be used, growth rates, extent of iron reduction, and the morphological changes that occur to minerals are poorly understood. The hyperthermophilic iron-reducing crenarchaea Pyrodictium delaneyi and Pyrobaculum islandicum were grown separately on six different synthetic nanophase Fe(III) (oxyhydr)oxides. For both organisms, growth on ferrihydrite produced the highest growth rates and the largest amounts of Fe(II), although P. delaneyi produced four times more Fe(II) (25 mM) than P. islandicum (6 mM). Both organisms grew on lepidocrocite and akaganéite and produced 2 and 3 mM Fe(II). Modest growth occurred for both organisms on goethite, hematite, and maghemite where ≤1 mM Fe(II) was produced. The diameters of the spherical mineral end-products following P. delaneyi growth increased by 30 nm for ferrihydrite and 50–150 nm for lepidocrocite relative to heated abiotic controls. For akaganéite, spherical particle sizes were the same for P. delaneyi-reacted samples and heated abiotic controls, but the spherical particles were more numerous in the P. delaneyi samples. For P. islandicum, there was no increase in grain size for the mineral end-products following growth on ferrihydrite, lepidocrocite, or akaganéite relative to the heated abiotic controls. High-resolution transmission electron microscopy of lattice fringes and selected-area electron diffraction of the minerals produced by both organisms when grown on ferrihydrite showed that magnetite and/or possibly maghemite were the end-products while the heated abiotic controls only contained ferrihydrite. These results expand the current view of bioavailable Fe(III) (oxyhydr)oxides for reduction by hyperthermophilic archaea when presented as synthetic nanophase minerals. They show that growth and reduction rates are inversely correlated with the iron (oxyhydr)oxide crystallinity and that iron (oxyhydr)oxide mineral transformation takes different forms for these two organisms.

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 the 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.

Thermococcus may be an important alternative source of H2 in the hot subseafloor in otherwise low H2 environments 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 H2 production. 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’s 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 < 1 mmol of H2 L-1. Twelve of 13 enzyme activities in T. paralvinellae showed significant (p