Deep-branching acetogens in serpentinized subsurface fluids of Oman.

Little is known of acetogens in contemporary serpentinizing systems, despite widely supported theories that serpentinite-hosted environments supported the first life on Earth via acetogenesis. To address this knowledge gap, genome-resolved metagenomics was applied to subsurface fracture water communities from an area of active serpentinization in the Samail Ophiolite, Sultanate of Oman. Two deeply branching putative bacterial acetogen types were identified in the communities belonging to the Acetothermia (hereafter, types I and II) that exhibited distinct distributions among waters with lower and higher water-rock reaction (i.e., serpentinization influence), respectively. Metabolic reconstructions revealed contrasting core metabolic pathways of type I and II Acetothermia, including in acetogenic pathway components (e.g., bacterial- vs. archaeal-like carbon monoxide dehydrogenases [CODH], respectively), hydrogen use to drive acetogenesis, and chemiosmotic potential generation via respiratory (type I) or canonical acetogen ferredoxin-based complexes (type II). Notably, type II Acetothermia metabolic pathways allow for use of serpentinization-derived substrates and implicate them as key primary producers in contemporary hyperalkaline serpentinite environments. Phylogenomic analyses indicate that 1) archaeal-like CODH of the type II genomes and those of other serpentinite-associated Bacteria derive from a deeply rooted horizontal transfer or origin among archaeal methanogens and 2) Acetothermia are among the earliest evolving bacterial lineages. The discovery of dominant and early-branching acetogens in subsurface waters of the largest near-surface serpentinite formation provides insight into the physiological traits that likely facilitated rock-supported life to flourish on a primitive Earth and possibly on other rocky planets undergoing serpentinization.

Living to Lithified: Construction and Preservation of Silicified Biomarkers.

Whole microorganisms are rarely preserved in the fossil record but actively silicifying environments like hot springs provide an opportunity for microbial preservation, making silicifying environments critical for the study of microbial life through time on Earth and possibly other planetary bodies. Yet, the changes that biosignatures may undergo through lithification and burial remain unconstrained. At Steep Cone Geyser in Yellowstone National Park, we collected microbial material from (1) the living system across the active outflows, (2) the silicified areas adjacent to flows, and (3) lithified and buried material to assess the preservation of biosignatures and their changes across the lithification transect. Five biofabrics, built predominantly by Cyanobacteria Geitlerinema, Pseudanabaenaceae, and Leptolyngbya with some filamentous anoxygenic phototrophs contributions, were identified and tracked from the living system through the process of silicification/lithification. In the living systems, δ30Si values decrease from +0.13‰ in surficial waters to -2‰ in biomat samples, indicating a kinetic isotope effect potentially induced by increased association with actively growing biofabrics. The fatty acids C16:1 and iso-C14:0 and the hydrocarbon C17:0 were disentangled from confounding signals and determined to be reliable lipid biosignatures for living biofabric builders and tenant microorganisms. Builder and tenant microbial biosignatures were linked to specific Cyanobacteria, anoxygenic phototrophs, and heterotrophs, which are prominent members of the living communities. Upon lithification and burial, silicon isotopes of silicified biomass began to re-equilibrate, increasing from δ30Si -2‰ in living biomats to -0.55‰ in lithified samples. Active endolithic microbial communities were identified in lithified samples and were dominated by Cyanobacteria, heterotrophic bacteria, and fungi. Results indicate that distinct microbial communities build and inhabit silicified biofabrics through time and that microbial biosignatures shift over the course of lithification. These findings improve our understanding of how microbial communities silicify, the biomarkers they retain, and transitionary impacts that may occur through lithification and burial.

Metapangenomic investigation provides insight into niche differentiation of methanogenic populations from the subsurface serpentinizing environment, Samail Ophiolite, Oman.

Serpentinization reactions produce highly reduced waters that have hyperalkaline pH and that can have high concentrations of H2 and CH4. Putatively autotrophic methanogenic archaea have been identified in the subsurface waters of the Samail Ophiolite, Sultanate of Oman, though the strategies to overcome hyperalkaline pH and dissolved inorganic carbon limitation remain to be fully understood. Here, we recovered metagenome assembled genomes (MAGs) and applied a metapangenomic approach to three different Methanobacterium populations to assess habitat-specific functional gene distribution. A Type I population was identified in the fluids with neutral pH, while a Type II and "Mixed" population were identified in the most hyperalkaline fluids (pH 11.63). The core genome of all Methanobacterium populations highlighted potential DNA scavenging techniques to overcome phosphate or nitrogen limitation induced by environmental conditions. With particular emphasis on the Mixed and Type II population found in the most hyperalkaline fluids, the accessory genomes unique to each population reflected adaptation mechanisms suggesting lifestyles that minimize niche overlap. In addition to previously reported metabolic capability to utilize formate as an electron donor and generate intracellular CO2, the Type II population possessed genes relevant to defense against antimicrobials and assimilating potential osmoprotectants to provide cellular stability. The accessory genome of the Mixed population was enriched in genes for multiple glycosyltransferases suggesting reduced energetic costs by adhering to mineral surfaces or to other microorganisms, and fostering a non-motile lifestyle. These results highlight the niche differentiation of distinct Methanobacterium populations to circumvent the challenges of serpentinization impacted fluids through coexistence strategies, supporting our ability to understand controls on methanogenic lifestyles and adaptations within the serpentinizing subsurface fluids of the Samail Ophiolite.

Spatial and Temporal Constraints on the Composition of Microbial Communities in Subsurface Boreholes of the Edgar Experimental Mine.

The deep biosphere hosts uniquely adapted microorganisms overcoming geochemical extremes at significant depths within the crust of the Earth. Attention is required to understand the near subsurface and its continuity with surface systems, where numerous novel microbial members with unique physiological modifications remain to be identified. This surface-subsurface relationship raises key questions about networking of surface hydrology, geochemistry affecting near-subsurface microbial composition, and resiliency of subsurface ecosystems. Here, we apply molecular and geochemical approaches to determine temporal microbial composition and environmental conditions of filtered borehole fluid from the Edgar Experimental Mine (∼150 m below the surface) in Idaho Springs, CO. Samples were collected over a 4-year collection period from expandable packers deployed to accumulate fluid in previously drilled boreholes located centimeters to meters apart, revealing temporal evolution of borehole microbiology. Meteoric groundwater feeding boreholes demonstrated variable recharge rates likely due to a complex and undefined fracture system within the host rock. 16S rRNA gene analysis determined that unique microbial communities occupy the four boreholes examined. Two boreholes yielded sequences revealing the presence of Desulfosporosinus, Candidatus Nitrotoga, and Chelatococcus associated with endemic subsurface communities. Two other boreholes presented sequences related to nonsubsurface-originating microbiota. High concentration of sulfate along with detected sulfur reducing and oxidizing microorganisms suggests that sulfur related metabolic strategies are prominent within these near-subsurface boreholes. Overall, results indicate that microbial community composition in the near-subsurface is highly dynamic at very fine spatial scales (<20 cm) within fluid-rock equilibrated boreholes, which additionally supports the role of a relationship for surface geochemical processes infiltrating and influencing subsurface environments. IMPORTANCE The Edgar Experimental Mine, Idaho Springs, CO, provides inexpensive and open access to borehole investigations for subsurface microbiology studies. Understanding how microbial processes in the near subsurface are connected to surface hydrological influences is lacking. Investigating microbial communities of subsurface mine boreholes provides evidence of how geochemical processes are linked to biogeochemical processes within each borehole and the geochemical connectedness and mobility of surface influences. This study details microbial community composition and fluid geochemistry over spatial and temporal scales from boreholes within the Edgar Mine. These findings are relevant to biogeochemistry of near-surface mines, caves, and other voids across planetary terrestrial systems. In addition, this work can lead to understanding how microbial communities relate to both fluid-rock equilibration, and geochemical influences may enhance our understanding of subsurface molecular biological tools that aid mining economic practices to reflect biological signals for lucrative veins in the near subsurface.