Microbial communities in terrestrial environments catalyze myriad biogeochemical cycles with global implications. However, in many ecosystems the combination of biological and chemical complexity can preclude a true mechanistic understanding of how microorganisms interact, are affected by viruses, and exchange metabolites. These kinds of insights are critical for our ability to manipulate microbiomes for beneficial outcomes, and better predict how biogeochemical cycles might change under varying environmental conditions. Deep fractured shales host more constrained microbial consortia that are more amenable to interrogation, and offer opportunities for new scientific insights and development of tools that can be applied to increasingly complex systems. Indeed, the investigator has previously revealed that the cycling of methylamines--simple carbon and nitrogen compounds--is a conserved metabolic strategy that fuels methylotrophic methanogenesis in deep shale formations across the Appalachian Basin. Work outlined in this proposal will determine the extent of functional conservation across geographically distinct shales, and use an extensive library of deep biosphere microbial isolates to investigate microbial metabolic interdependencies that enable persistence of life in these extreme environments. The role of viruses in driving microbial community dynamics and metabolite cycling will also be investigated. Finally, novel tools and insights developed in the shale ecosystem will be applied to a more complex wetland system to further illuminate the extent of methylamine metabolism in terrestrial environments. This project will train a graduate student and offer opportunities to undergraduate researchers. The involvement of industrial partners will enable internship placements for graduate students, and contribute to curriculum materials aimed at informing students of diverse career opportunities in subsurface science. Given the level of public interest in hydraulic fracturing and deep biosphere topics, a series of outreach events at local organizations are also planned.<br/><br/>Genome-resolved metagenomic and metabolomic analyses, coupled with laboratory experimentation, has revealed that methylamine cycling in deep terrestrial shales is a conserved functional process across the Appalachian Basin that enables microbial communities to persist for many hundreds of days. Using multiomic approaches, this work will expand investigations into western US shale plays, and determine how functional traits are altered under varying salinity and temperature conditions. Genomic insights into metabolite exchange and methane isotope fractionation in these systems will be tested in the laboratory using environmental isolates and high-pressure incubation apparatus that enables in situ conditions (e.g., pressure, temperature, salinity) to be recreated. Novel analytical tools including high-pressure real-time NMR will be used to track the cycling of substrates at high temporal resolution. The ability of novel and abundant viral populations to mediate carbon and nitrogen cycling in the deep biosphere will also be investigated using a combination of bioinformatic tools and laboratory experimentation. Data resulting from these studies will offer unique insights into the functioning of microbial and viral populations, and the impact of microbial metabolism on poorly-resolved carbon and nitrogen cycles in the deep terrestrial subsurface.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.