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Deep marine sediments (>1mbsf) harbor ~26% of microbial biomass and are the largest reservoir of methane on Earth. Yet, the deep subsurface biosphere and controls on its contribution to methane production remain underexplored. Here, we use a multidisciplinary approach to examine methanogenesis in sediments (down to 295 mbsf) from sites with varying degrees of thermal alteration (none, past, current) at Guaymas Basin (Gulf of California) for the first time. Traditional (13C/12C and D/H) and multiply substituted (13CH3D and 12CH2D2) methane isotope measurements reveal significant proportions of microbial methane at all sites, with the largest signal at the site with past alteration. With depth, relative microbial methane decreases at differing rates between sites. Gibbs energy calculations confirm methanogenesis is exergonic in Guaymas sediments, with methylotrophic pathways consistently yielding more energy than the canonical hydrogenotrophic and acetoclastic pathways. Yet, metagenomic sequencing and cultivation attempts indicate that methanogens are present in low abundance. We find only one methyl-coenzyme M (mcrA) sequence within the entire sequencing dataset. Also, we identify a wide diversity of methyltransferases (mtaB, mttB), but only a few sequences phylogenetically cluster with methylotrophic methanogens. Our results suggest that the microbial methane in the Guaymas subsurface was produced over geologic time by relatively small methanogen populations, which have been variably influenced by thermal sediment alteration. Higher resolution metagenomic sampling may clarify the modern methanogen community. This study highlights the importance of using a multidisciplinary approach to capture microbial influences in dynamic, deep subsurface settings like Guaymas Basin.

 

Biogeochemical sulfur cycling in sulfidic karst systems is largely driven by abiotic and biological sulfide oxidation, but the fate of elemental sulfur (S⁰) that accumulates in these systems is not well understood. The Frasassi Cave system (Italy) is intersected by a sulfidic aquifer that mixes with small quantities of oxygen‐rich meteoric water, creating Proterozoic‐like conditions and supporting a prolific ecosystem driven by sulfur‐based chemolithoautotrophy. To better understand the cycling of S⁰in this environment, we examined the geochemistry and microbiology of sediments underlying widespread sulfide‐oxidizing mats dominated byBeggiatoa. Sediment populations were dominated by uncultivated relatives of sulfur cycling chemolithoautotrophs related toSulfurovum,Halothiobacillus,Thiofaba,Thiovirga,Thiobacillus, andDesulfocapsa, as well as diverse uncultivated anaerobic heterotrophs affiliated withBacteroidota, Anaerolineaceae, Lentimicrobiaceae, and Prolixibacteraceae.DesulfocapsaandSulfurovumpopulations accounted for 12%–26% of sediment 16S rRNA amplicon sequences and were closely related to isolates which carry out autotrophic S⁰disproportionation in pure culture. Gibbs energy (∆G_r) calculations revealed that S⁰disproportionation under in situ conditions is energy yielding. Microsensor profiles through the mat‐sediment interface showed thatBeggiatoamats consume dissolved sulfide and oxygen, but a net increase in acidity was only observed in the sediments below. Together, these findings suggest that disproportionation is an important sink for S⁰generated by microbial sulfide oxidation in this oxygen‐limited system and may contribute to the weathering of carbonate rocks and sediments in sulfur‐rich environments.

 

The microbial ecology of the deep biosphere is difficult to characterize, owing in part to sampling challenges and poorly understood response mechanisms to environmental change. Pre-drilled wells, including oil wells or boreholes, offer convenient access, but sampling is frequently limited to the water alone, which may provide only a partial view of the native diversity. Mineral heterogeneity demonstrably affects colonization by deep biosphere microorganisms, but the connections between the mineral-associated and planktonic communities remain unclear. To understand the substrate effects on microbial colonization and the community response to changes in organic carbon, we conducted an 18-month series of in situ experiments in a warm (57°C), anoxic, fractured carbonate aquifer at 752 m depth using replicate open, screened cartridges containing different solid substrates, with a proteinaceous organic matter perturbation halfway through this series. Samples from these cartridges were analyzed microscopically and by Illumina (iTag) 16S rRNA gene libraries to characterize changes in mineralogy and the diversity of the colonizing microbial community. The substrate-attached and planktonic communities were significantly different in our data, with some taxa (e.g., Candidate Division KB-1) rare or undetectable in the first fraction and abundant in the other. The substrate-attached community composition also varied significantly with mineralogy, such as with two Rhodocyclaceae OTUs, one of which was abundant on carbonate minerals and the other on silicic substrates. Secondary sulfide mineral formation, including iron sulfide framboids, was observed on two sets of incubated carbonates. Notably, microorganisms were attached to the framboids, which were correlated with abundant Sulfurovum and Desulfotomaculum sp. sequences in our analysis. Upon organic matter perturbation, mineral-associated microbial diversity differences were temporarily masked by the dominance of putative heterotrophic taxa in all samples, including OTUs identified as Caulobacter , Methyloversatilis , and Pseudomonas . Subsequent experimental deployments included a methanogen-dominated stage ( Methanobacteriales and Methanomicrobiales ) 6 months after the perturbation and a return to an assemblage similar to the pre-perturbation community after 9 months. Substrate-associated community differences were again significant within these subsequent phases, however, demonstrating the value of in situ time course experiments to capture a fraction of the microbial assemblage that is frequently difficult to observe in pre-drilled wells. 

The methyl-coenzyme M reductase (MCR) complex is a key enzyme in archaeal methane generation and has recently been proposed to also be involved in the oxidation of short-chain hydrocarbons including methane, butane, and potentially propane. The number of archaeal clades encoding the MCR continues to grow, suggesting that this complex was inherited from an ancient ancestor, or has undergone extensive horizontal gene transfer. Expanding the representation of MCR-encoding lineages through metagenomic approaches will help resolve the evolutionary history of this complex. Here, a near-complete Archaeoglobi metagenome-assembled genome (MAG; Ca. Polytropus marinifundus gen. nov. sp. nov.) was recovered from the deep subseafloor along the Juan de Fuca Ridge flank that encodes two divergent McrABG operons similar to those found in Ca. Bathyarchaeota and Ca. Syntrophoarchaeum MAGs. Ca. P. marinifundus is basal to members of the class Archaeoglobi, and encodes the genes for β-oxidation, potentially allowing an alkanotrophic metabolism similar to that proposed for Ca. Syntrophoarchaeum. Ca. P. marinifundus also encodes a respiratory electron transport chain that can potentially utilize nitrate, iron, and sulfur compounds as electron acceptors. Phylogenetic analysis suggests that the Ca. P. marinifundus MCR operons were horizontally transferred, changing our understanding of the evolution and distribution of this complex in the Archaea.

 

The biology literature is rife with misleading information on how to quantify catabolic reaction energetics. The principal misconception is that the sign and value of thestandardGibbs energy () define the direction and energy yield of a reaction; they do not.is one part of theactualGibbs energy of a reaction (ΔG_r), with a second part accounting for deviations from the standard composition. It is also frequently assumed thatapplies only to 25 °C and 1 bar; it does not.is a function of temperature and pressure. Here, we review how to determineΔG_ras a function of temperature, pressure and chemical composition for microbial catabolic reactions, including a discussion of the effects of ionic strength onΔG_rand highlighting the large effects when multi‐valent ions are part of the reaction. We also calculateΔG_rfor five example catabolisms at specific environmental conditions: aerobic respiration of glucose in freshwater, anaerobic respiration of acetate in marine sediment, hydrogenotrophic methanogenesis in a laboratory batch reactor, anaerobic ammonia oxidation in a wastewater reactor and aerobic pyrite oxidation in acid mine drainage. These examples serve as templates to determine the energy yields of other catabolic reactions at environmentally relevant conditions.

 

Chemotrophic microorganisms gain energy for cellular functions by catalyzing oxidation–reduction (redox) reactions that are out of equilibrium. Calculations of the Gibbs energy (ΔG_r) can identify whether a reaction is thermodynamically favourable and quantify the accompanying energy yield at the temperature, pressure and chemical composition in the system of interest. Based on carefully calculated values ofΔG_r, we predict a novel microbial metabolism – sulfur comproportionation (3H₂S ++ 2H⁺⇌4S⁰+ 4H₂O). We show that at elevated concentrations of sulfide and sulfate in acidic environments over a broad temperature range, this putative metabolism can be exergonic (ΔG_r<0), yielding ~30–50 kJ mol⁻¹. We suggest that this may be sufficient energy to support a chemolithotrophic metabolism currently missing from the literature. Other versions of this metabolism, comproportionation to thiosulfate (H₂S +⇌+ H₂O) and to sulfite (H₂S + 3⇌4+ 2H⁺), are only moderately exergonic or endergonic even at ideal geochemical conditions. Natural and impacted environments, including sulfidic karst systems, shallow‐sea hydrothermal vents, sites of acid mine drainage, and acid–sulfate crater lakes, may be ideal hunting grounds for finding microbial sulfur comproportionators.