ONE OF THE BIGGEST CHALLENGES IN THE TRANSLATION OF ENVIRONMENTAL MICROBIOLOGY RESEARCH TO UNDERSTANDING EARLY-LIFE AND THE COEVOLUTION OF LIFE AND THE ENVIRONMENT IS UNTANGLING THE FUNCTIONAL POTENTIAL OF THE VAST SUITE OF UNCULTIVATED MICROORGANISMS THAT REPRESENT THE MAJORITY OF MICROBIAL COMMUNITIES. WHILE RECENT ADVANCES IN DNA SEQUENCING METAGENOMIC AND SINGLE CELL GENOMIC TECHNIQUES HAVE REVOLUTIONIZED THE ABILITY TO UNCOVER GENOMIC INFORMATION FROM THESE GROUPS LINKAGE OF THIS INFORMATION TO FUNCTION IN THE ENVIRONMENT IS TENUOUS. THIS CHALLENGE IS MAGNIFIED IN LOW-ENERGY ANALOG ENVIRONMENTS WHERE MEASURING RATES OF ACTIVITY IS DIFFICULT DUE TO DETECTION LIMITS. THE GOAL OF THIS PROJECT IS TO TACKLE THIS CHALLENGE THROUGH APPLICATION OF NASCENT SINGLE-CELL-LEVEL FLUORESCENT SUBSTRATE LABELING TECHNIQUES THAT IDENTIFY ACTIVE CELLS USING FLOW CYTOMETRIC SORTING AND GENOMIC SEQUENCING IN A RELEVANT ANALOG SYSTEM. THE PROJECT WILL TAKE ADVANTAGE OF SAMPLES-OF-OPPORTUNITY TO BE COLLECTED FROM THE SUBSURFACE ENVIRONMENT OF EARTH S OCEANIC CRUST (AND ICY WORLDS ANALOG) WHERE FLUID-ROCK REACTIONS IN MAFIC ROCKS SUPPORT AN ENERGY-LIMITED CHEMOLITHOTROPHIC ECOSYSTEM. SPECIFICALLY THIS PROJECT WILL LEVERAGE AN NSF-FUNDED EXPEDITION IN 2019 TO SUBSEAFLOOR OBSERVATORIES ON THE EASTERN FLANK OF THE JUAN DE FUCA RIDGE TO ENABLE IN SITU EXPERIMENTATION AND COLLECTION OF PRISTINE SAMPLES. LIVE MICROBIAL CELLS IN THIS HIGH-PRESSURE THERMOPHILIC (64 C) ANOXIC SYSTEM WILL BE SELECTED THROUGH INCORPORATION OF FLUOROGENIC SUBSTRATES INSIDE THE CELL ENABLING SORTING OF THESE CELLS FROM THE BULK COMMUNITY THROUGH FLOW CYTOMETRY. VARIOUS METABOLISMS INCLUDING CARBON MONOXIDE IRON AND SULFUR OXIDATION WILL BE EXPLORED THROUGH ENRICHMENT EXPERIMENTS PATHWAYS OF RELEVANCE TO CONSTRAINING CHEMOLITHOTROPHY ON EXTRATERRESTRIAL TARGETS LIKE EUROPA AND ENCELADUS. PRELIMINARY EXPERIMENTS CONDUCTED WITH SIMILAR SUBSURFACE OCEAN CRUST SAMPLES FROM A DIFFERENT SYSTEM CONFIRM THAT CELLS IN LOW BIOMASS LOW ENERGY SYSTEMS CAN BE LABELED IN THIS WAY AND THAT THESE CELLS ARE AMENABLE TO DOWNSTREAM GENOME SEQUENCING. IF SUCCESSFUL RESULTS OF THIS PROJECT WILL SIGNIFICANTLY ADVANCE UNDERSTANDING OF THE SPECIFIC FUNCTIONAL POTENTIAL AND VIABILITY OF VARIOUS MEMBERS OF LOW-ENERGY SUBSURFACE ANALOG MICROBIAL COMMUNITIES REGARDLESS OF WHETHER THEY ARE DOMINANT OR RARE MEMBERS OF THE POPULATION. THIS WOULD ESTABLISH A REVOLUTIONARY APPROACH TO CONNECTING PHYSIOLOGY TO GENOMIC INFORMATION WHICH HAS APPLICATIONS BEYOND EXOBIOLOGY. CONFIRMING THE APPLICATION OF THIS METHOD IN A REMOTE ANALOG SYSTEM WOULD IMPACT NASA MISSION INTERESTS SEEKING TO IDENTIFY METHODS FOR BIOSIGNATURE DETECTION AND POSSIBLE SCENARIOS FOR EXPERIMENTATION ON FUTURE ROVERS TO IDENTIFY LIFE. THE PROPOSED PROJECT IS RELEVANT TO SPECIFIC RESEARCH EMPHASES IN THIS SOLICITATION AS WELL AS SEVERAL OF THE MAJOR TOPICS IN THE NASA ASTROBIOLOGY STRATEGY 2015. BY SORTING ACTIVE CELLS FROM OCEAN CRUST ANALOG ENVIRONMENTS AND THEN BEING ABLE TO DIRECTLY LINK CELL PHYSIOLOGY WITH GENOMIC INFORMATION THIS PROJECT DIRECTLY ADDRESSES THE SOLICITATION INTERESTS IN UNDERSTANDING THE PHYLOGENY AND PHYSIOLOGY OF EXTREMOPHILE MICROORGANISMS THAT MAY REFLECT THE NATURE OF PRIMITIVE ENVIRONMENTS AND THE CO-EVOLUTION OF THESE MICROBIAL COMMUNITIES THAT DRIVE THE BIOGEOCHEMICAL PROCESSES IN THESE ANALOG ENVIRONMENTS WHICH IS LIKEWISE DIRECTLY RELEVANT TO THEMES IN THE MAJOR TOPIC CO-EVOLUTION OF LIFE AND THE PHYSICAL ENVIRONMENT . MOREOVER BY FOCUSING ON ENERGY LIMITED ANALOG SYSTEMS RELEVANT TO NASA S INTEREST IN ICY WORLDS THIS RESEARCH DIRECTLY CONTRIBUTES TO THEMES IN THE MAJOR TOPIC EARLY LIFE AND INCREASING COMPLEXITY.