Genomic Description of ‘Candidatus Abyssubacteria,’ a Novel Subsurface Lineage Within the Candidate Phylum Hydrogenedentes

The subsurface biosphere is a massive repository of fixed carbon, harboring approximately 90% of Earth’s microbial biomass. These microbial communities drive transformations central to Earth’s biogeochemical cycles. However, there is still much we do not understand about how complex subterranean microbial communities survive and how they interact with these cycles. Recent metagenomic investigation of deeply circulating terrestrial subsurface fluids revealed the presence of several novel lineages of bacteria. In one particular example, phylogenomic analyses do not converge on any one previously identified taxon; here we describe the first full genomic sequences of a new bacterial lineage within the candidate phylum Hydrogenedentes, ‘Candidatus Abyssubacteria.’ A global survey revealed that members of this proposed lineage are widely distributed in both marine and terrestrial subsurface environments, but their physiological and ecological roles have remained unexplored. Two high quality metagenome assembled genomes (SURF_5: 97%, 4%; SURF_17: 91% and 4% completeness and contamination, respectively) were reconstructed from fluids collected 1.5 kilometers below surface in the former Homestake gold mine—now the Sanford Underground Research Facility (SURF)—in Lead, South Dakota, United States. Metabolic reconstruction suggests versatile metabolic capability, including possible nitrogen reduction, sulfite oxidation, sulfate reduction and homoacetogenesis. This first glimpse into the metabolic capabilities of these cosmopolitan bacteria suggests that they are involved in key geochemical processes, including sulfur, nitrogen, and carbon cycling, and that they are adapted to survival in the dark, often anoxic, subsurface biosphere

The Energetic Potential for Undiscovered Manganese Metabolisms in Nature

Microorganisms are found in nearly every surface and near-surface environment, where they gain energy by catalyzing reactions among a wide variety of chemical compounds. The discovery of new catabolic strategies and microbial habitats can therefore be guided by determining which redox reactions can supply energy under environmentally-relevant conditions. In this study, we have explored the thermodynamic potential of redox reactions involving manganese, one of the most abundant transition metals in the Earth’s crust. In particular, we have assessed the Gibbs energies of comproportionation and disproportionation reactions involving Mn2+ and several Mn-bearing oxide and oxyhydroxide minerals containing Mn in the +II, +III, and +IV oxidation states as a function of temperature (0–100°C) and pH (1–13). In addition, we also calculated the energetic potential of Mn2+ oxidation coupled to O2, NO2–, NO3–, and FeOOH. Results show that these reactions—none of which, except O2 + Mn2+, are known catabolisms—can provide energy to microorganisms, particularly at higher pH values and temperatures. Comproportionation between Mn2+ and pyrolusite, for example, can yield 10 s of kJ (mol Mn)–1. Disproportionation of Mn3+ can yield more than 100 kJ (mol Mn)–1 at conditions relevant to natural settings such as sediments, ferromanganese nodules and crusts, bioreactors and suboxic portions of the water column. Of the Mn2+ oxidation reactions, the one with nitrite as the electron acceptor is most energy yielding under most combinations of pH and temperature. We posit that several Mn redox reactions represent heretofore unknown microbial metabolisms

Sulfur and oxygen isotope insights into sulfur cycling in shallow-sea hydrothermal vents, Milos, Greece

Shallow-sea (5 m depth) hydrothermal venting off Milos Island provides an ideal opportunity to target transitions between igneous abiogenic sulfide inputs and biogenic sulfide production during microbial sulfate reduction. Seafloor vent features include large (>1 m2) white patches containing hydrothermal minerals (elemental sulfur and orange/yellow patches of arsenic-sulfides) and cells of sulfur oxidizing and reducing microorganisms. Sulfide-sensitive film deployed in the vent and non-vent sediments captured strong geochemical spatial patterns that varied from advective to diffusive sulfide transport from the subsurface. Despite clear visual evidence for the close association of vent organisms and hydrothermalism, the sulfur and oxygen isotope composition of pore fluids did not permit delineation of a biotic signal separate from an abiotic signal. Hydrogen sulfide (H2S) in the free gas had uniform δ34S values (2.5 ± 0.28‰, n = 4) that were nearly identical to pore water H2S (2.7 ± 0.36‰, n = 21). In pore water sulfate, there were no paired increases in δ34SSO4 and δ18OSO4 as expected of microbial sulfate reduction. Instead, pore water δ34SSO4 values decreased (from approximately 21‰ to 17‰) as temperature increased (up to 97.4°C) across each hydrothermal feature. We interpret the inverse relationship between temperature and δ34SSO4 as a mixing process between oxic seawater and 34S-depleted hydrothermal inputs that are oxidized during seawater entrainment. An isotope mass balance model suggests secondary sulfate from sulfide oxidation provides at least 15% of the bulk sulfate pool. Coincident with this trend in δ34SSO4, the oxygen isotope composition of sulfate tended to be 18O-enriched in low pH (75°C) pore waters. The shift toward high δ18OSO4 is consistent with equilibrium isotope exchange under acidic and high temperature conditions. The source of H2S contained in hydrothermal fluids could not be determined with the present dataset; however, the end-member δ34S value of H2S discharged to the seafloor is consistent with equilibrium isotope exchange with subsurface anhydrite veins at a temperature of ~300°C. Any biological sulfur cycling within these hydrothermal systems is masked by abiotic chemical reactions driven by mixing between low-sulfate, H2S-rich hydrothermal fluids and oxic, sulfate-rich seawater

Sulfur disproportionating microbial communities in a dynamic, microoxic‐sulfidic karst system

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 S0 in this environment, we examined the geochemistry and microbiology of sediments underlying widespread sulfide-oxidizing mats dominated by Beggiatoa. Sediment populations were dominated by uncultivated relatives of sulfur cycling chemolithoautotrophs related to Sulfurovum, Halothiobacillus, Thiofaba, Thiovirga, Thiobacillus, and Desulfocapsa, as well as diverse uncultivated anaerobic heterotrophs affiliated with Bacteroidota, Anaerolineaceae, Lentimicrobiaceae, and Prolixibacteraceae. Desulfocapsa and Sulfurovum populations accounted for 12%–26% of sediment 16S rRNA amplicon sequences and were closely related to isolates which carry out autotrophic S0 disproportionation in pure culture. Gibbs energy (∆Gr) calculations revealed that S0 disproportionation under in situ conditions is energy yielding. Microsensor profiles through the mat-sediment interface showed that Beggiatoa mats 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 S0 generated by microbial sulfide oxidation in this oxygen-limited system and may contribute to the weathering of carbonate rocks and sediments in sulfur-rich environments

Patterns of in situ Mineral Colonization by Microorganisms in a ~60°C Deep Continental Subsurface Aquifer

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

Spatially and temporally variable sulfur cycling in shallow-sea hydrothermal vents, Milos, Greece

Shallow-sea hydrothermal systems are ideal for studying the relative contributions to sedimentary sulfur archives from ambient sulfur-utilizing microbes and from fluxes of hydrothermally derived sulfur. Here we present data from a vent field in Palaeochori Bay, Milos, Greece using a suite of biogeochemical analytical tools that captured both spatial and temporal variability in biotic and abiotic sulfur cycling. Samples were collected along a transect from a seagrass meadow to an area of active venting. The abundance and isotopic composition of sulfide captured in situ, together with geochemistry from sedimentary porewaters and the overlying water column and solid phase sulfide minerals, record evidence of ephemeral activity of microbial sulfate reduction as well as sulfide oxidation. The sulfur and oxygen isotope composition of porewater sulfates indicate active sulfate reduction within the transition zone between the vents and seagrass, rapid recycling of biologically produced sulfide within non-vent sediments, and reoxidation of abiotic sulfide within the vent field. A phylogenetic survey of sediments also indicates the pervasive presence of a suite of putative sulfur-metabolizing bacteria, including sulfate reducers and sulfide oxidizers, many of which can utilize intermediate valence sulfur compounds. The isotopic composition of pyrite in these sediments consistently records a microbially influenced signature (δ34Spy of −4.4 to −10.8‰) relative to the hydrothermal endmember (δ34S ~ + 2.5‰), independent of distance from the vent source. The narrow range of pyrite δ34S across sediments with a highly variable hydrothermal influence suggests that physical mixing (e.g., by storm events) homogenizes the distribution of biogenic and hydrothermal Fe-sulfides throughout the region, overprinting the spatially and temporally variable interplay between biological and hydrothermal sulfur cycling in these environments

Subsurface hydrothermal processes and the bioenergetics of chemolithoautotrophy at the shallow-sea vents off Panarea Island (Italy)

The subsurface evolution of shallow-sea hydrothermal fluids is a function of many factors including fluid-mineral equilibria, phase separation, magmatic inputs, and mineral precipitation, all of which influence discharging fluid chemistry and consequently associated seafloor microbial communities. Shallow-sea vent systems, however, are understudied in this regard. In order to investigate subsurface processes in a shallow-sea hydrothermal vent, and determine how these physical and chemical parameters influence the metabolic potential of the microbial communities, three shallow-sea hydrothermal vents associated with Panarea Island (Italy) were characterized. Vent fluids, pore fluids and gases at the three sites were sampled and analyzed for major and minor elements, redox-sensitive compounds, free gas compositions, and strontium isotopes. The corresponding data were used to 1) describe the subsurface geochemical evolution of the fluids and 2) to evaluate the catabolic potential of 61 inorganic redox reactions for in situ microbial communities. Generally, the vent fluids can be hot (up to 135 °C), acidic (pH 1.9-5.7), and sulfidic (up to 2.5 mM H2S). Three distinct types of hydrothermal fluids were identified, each with higher temperatures and lower pH, Mg2 + and SO42 -, relative to seawater. Type 1 was consistently more saline than Type 2, and both were more saline than seawater. Type 3 fluids were similar to or slightly depleted in most major ions relative to seawater. End-member calculations of conservative elements indicate that Type 1 and Type 2 fluids are derived from two different sources, most likely 1) a deeper, higher salinity reservoir and 2) a shallower, lower salinity reservoir, respectively, in a layered hydrothermal system. The deeper reservoir records some of the highest end-member Cl concentrations to date, and developed as a result of recirculation of brine fluids with long term loss of steam and volatiles due to past phase separation. No strong evidence for ongoing phase separation is observed. Type 3 fluids are suggested to be mostly influenced by degassing of volatiles and subsequently dissolution of CO2, H2S, and other gases into the aqueous phase. Gibbs energies (ΔGr) of redox reactions that couple potential terminal electron acceptors (O2, NO3-, MnIV, FeIII, SO42 -, S0, CO2,) with potential electron donors (H2, NH4+, Fe2 +, Mn2 +, H2S, CH4) were evaluated at in situ temperatures and compositions for each site and by fluid type. When Gibbs energies of reaction are normalized per kilogram of hydrothermal fluid, sulfur oxidation reactions are the most exergonic, while the oxidation of Fe2 +, NH4+, CH4, and Mn2 + are moderately energy yielding. The energetics calculations indicate that the most robust microbial communities in the Panarea hot springs combine H2S from deep water-rock-gas interactions with O2 that is entrained via seawater mixing to fuel their activities, regardless of site location or fluid type