A metagenomic view of novel microbial and metabolic diversity found within the deep terrestrial biosphere at DeMMO: A microbial observatory in South Dakota, USA.

The deep terrestrial subsurface is a large and diverse microbial habitat and vast repository of biomass. However, in relation to its size and physical heterogeneity we have limited understanding of taxonomic and metabolic diversity in this realm. Here we present a detailed metagenomic analysis of samples from the Deep Mine Microbial Observatory (DeMMO) spanning depths from the surface to 1.5 km into the crust. From eight geochemically and spatially distinct fluid samples we reconstructed ~600 partial to near-complete metagenome-assembled genomes (MAGs), representing 50 distinct phyla and including 18 candidate phyla. These novel clades include members of the candidate phyla radiation, two new MAGs from OLB16, a phylum originally identified in DeMMO fluids and for which only one other MAG is currently available, and new MAGs from the Eisenbacteria, Omnitrophota, and Edwardsbacteria. We find that microbes spanning this expansive phylogenetic diversity and physical subsurface space gain a competitive edge by maintaining a wide variety of functional pathways, are often capable of numerous dissimilatory energy metabolisms and poised to take advantage of nutrients as they become available in isolated fracture fluids. Our results support and expand on emerging themes of tight nutrient cycling and genomic plasticity in deep subsurface biosphere taxa.

Rock-Hosted Subsurface Biofilms: Mineral Selectivity Drives Hotspots for Intraterrestrial Life.

The continental deep subsurface is likely the largest reservoir of biofilm-based microbial biomass on Earth, but the role of mineral selectivity in regulating its distribution and diversity is unclear. Minerals can produce hotspots for intraterrestrial life by locally enhancing biofilm biomass. Metabolic transformations of minerals by subsurface biofilms may occur widely with the potential to significantly impact subsurface biogeochemical cycles. However, the degree of impact depends upon the amount of biofilm biomass and its relationship to host rock mineralogy, estimates that are currently loosely constrained to non-existent. Here, we use in situ cultivation of biofilms on native rocks and coupled microscopy/spectroscopy to constrain mineral selectivity by biofilms in a deep continental subsurface setting: the Deep Mine Microbial Observatory (DeMMO). Through hotspot analysis and spatial modeling approaches we find that mineral distributions, particularly those putatively metabolized by microbes, indeed drive biofilm distribution at DeMMO, and that bioleaching of pyrite may be a volumetrically important process influencing fluid geochemistry at this site when considered at the kilometer scale. Given the ubiquity of iron-bearing minerals at this site and globally, and the amount of biomass they can support, we posit that rock-hosted biofilms likely contribute significantly to subsurface biogeochemical cycles. As more data becomes available, future efforts to estimate biomass in the continental subsurface should incorporate host rock mineralogy.

Electrochemical evidence for in situ microbial activity at the Deep Mine Microbial Observatory (DeMMO), South Dakota, USA.

The subsurface is Earth's largest reservoir of biomass. Micro-organisms are the dominant lifeforms in this habitat, but the nature of their in situ activities remains largely unresolved. At the Deep Mine Microbial Observatory (DeMMO) located in the Sanford Underground Research Facility (SURF) in Lead, South Dakota (USA), we performed in situ electrochemical incubations designed to assess the potential for deep groundwater microbial communities to utilize extracellular electron transfer to support microbial respiration. DeMMO 4 was chosen for its stable geochemistry and microbial community. Graphite and indium tin oxide electrodes poised at -200 mV versus SHE were incubated along with open circuit controls and various minerals in a parallel flow reactor that split access to fluids across different treatments. From the patterns of net current over time (fluctuating between anodic and cathodic currents over the course of a few days to weeks) and the catalytic features measured using periodic cyclic voltammetry, evidence of both oxidative and reductive microbe-electrode interactions was observed. The predominant catalytic activity ranged from -210 to -120 mV. The observed temporal variability in electrochemical activity was unexpected given the documented stability in major geochemical parameters. This suggests that the accessed fluids are more heterogeneous in electrochemically active microbial populations than previously predicted from the stable community composition. As previously reported, the fracture fluid and surface-attached microbial communities at SURF differed significantly. However, only minimal differences in community composition were observed between poised potential electrodes, open circuit electrodes, and mineral incubations. These data support that in this environment the ability to attach to surfaces is a stronger driver of microbial community structure than the type or reactivity of the surface. We demonstrate that insight into specific activities can be gained from electrochemical methods, specifically chronoamperometry coupled with routine cyclic voltammetry, which provide a sensitive approach to evaluate microbial activities in situ.

Mineral-hosted biofilm communities in the continental deep subsurface, Deep Mine Microbial Observatory, SD, USA.

Deep subsurface biofilms are estimated to host the majority of prokaryotic life on Earth, yet fundamental aspects of their ecology remain unknown. An inherent difficulty in studying subsurface biofilms is that of sample acquisition. While samples from marine and terrestrial deep subsurface fluids have revealed abundant and diverse microbial life, limited work has described the corresponding biofilms on rock fracture and pore space surfaces. The recently established Deep Mine Microbial Observatory (DeMMO) is a long-term monitoring network at which we can explore the ecological role of biofilms in fluid-filled fractures to depths of 1.5 km. We carried out in situ cultivation experiments with single minerals representative of DeMMO host rock to explore the ecological drivers of biodiversity and biomass in biofilm communities in the continental subsurface. Coupling cell densities to thermodynamic models of putative metabolic reactions with minerals suggests a metabolic relationship between biofilms and the minerals they colonize. Our findings indicate that minerals can significantly enhance biofilm cell densities and promote selective colonization by taxa putatively capable of extracellular electron transfer. In turn, minerals can drive significant differences in biodiversity between fluid and biofilm communities. Given our findings at DeMMO, we suggest that host rock mineralogy is an important ecological driver in deep continental biospheres.

Biofilm formation and potential for iron cycling in serpentinization-influenced groundwater of the Zambales and Coast Range ophiolites.

Terrestrial serpentinizing systems harbor microbial subsurface life. Passive or active microbially mediated iron transformations at alkaline conditions in deep biosphere serpentinizing ecosystems are understudied. We explore these processes in the Zambales (Philippines) and Coast Range (CA, USA) ophiolites, and associated surface ecosystems by probing the relevance of samples acquired at the surface to in situ, subsurface ecosystems, and the nature of microbe-mineral associations in the subsurface. In this pilot study, we use microcosm experiments and batch culturing directed at iron redox transformations to confirm thermodynamically based predictions that iron transformations may be important in subsurface serpentinizing ecosystems. Biofilms formed on rock cores from the Zambales ophiolite on surface and in-pit associations, confirming that organisms from serpentinizing systems can form biofilms in subsurface environments. Analysis by XPS and FTIR confirmed that enrichment culturing utilizing ferric iron growth substrates produced reduced, magnetic solids containing siderite, spinels, and FeO minerals. Microcosms and enrichment cultures supported organisms whose near relatives participate in iron redox transformations. Further, a potential 'principal' microbial community common to solid samples in serpentinizing systems was identified. These results indicate collectively that iron redox transformations should be more thoroughly and universally considered when assessing the function of terrestrial subsurface ecosystems driven by serpentinization.