ABSTRACT: Deep subsurface environments are decoupled from Earth's surface processes yet diverse, active, and abundant microbial communities thrive in these isolated environments. Microbes inhabiting the deep biosphere face unique challenges such as electron donor/acceptor limitations, pore space/fracture network limitations, and isolation from other microbes within the formation. Of the few systems that have been characterized, it is apparent that nutrient limitations likely facilitate diverse microbe-microbe interactions (i.e., syntrophic, symbiotic, or parasitic) and that these interactions drive biogeochemical cycling of major elements. Here we describe microbial communities living in low temperature, chemically reduced brines at the Soudan Underground Mine State Park, United States. The Soudan Iron mine intersects a massive hematite formation at the southern extent of the Canadian Shield. Fractured rock aquifer brines continuously flow from exploratory boreholes drilled circa 1960 and are enriched in deuterium compared to the global meteoric values, indicating brines have had little contact with surface derived waters, and continually degas low molecular weight hydrocarbons C1-C4. Microbial enrichments suggest that once brines exit the boreholes, oxidation of the hydrocarbons occur. Amplicon sequencing show these borehole communities are low in diversity and dominated by Firmicute and Proteobacteria phyla. From the metagenome assemblies, we recovered approximately thirty genomes with estimated completion over 50%. Analysis of genome taxonomy generally followed the amplicon data, and highlights that several of the genomes represent novel families and genera. Metabolic reconstruction shows two carbon-fixation pathways were dominant, the Wood-Ljungdahl (acetogenesis) and Calvin-Benson-Bassham (via RuBisCo), indicating that inorganic carbon likely enters into the microbial foodweb with differing carbon fractionation potentials. Interestingly, methanogenesis is likely driven by Methanolobus and suggests cycling of methylated compounds and not H2/CO2 or acetate. Furthermore, the abundance of sulfate in brines suggests cryptic sulfur cycling may occur, as we detect possible sulfate reducing and thiosulfate oxidizing microorganisms. Finally, a majority of the microorganisms identified contain genes that would allow them to participate in several element cycles, highlighting that in these deep isolated systems metabolic flexibility may be an important life history trait.
