HUMAN MISSIONS TO MARS ARE NOW UNDER ACTIVE PLANNING AT NASA. IN ORDER TO DEVELOP HARDWARE SPACESUITS AND HABITATS THAT MEET MISSION OBJECTIVES WHILE ALSO PREVENTING FORWARD CONTAMINATION THE SURVIVAL METABOLISM AND GROWTH OF TERRESTRIAL MICROORGANISMS UNDER MARS SURFACE CONDITIONS MUST BE CHARACTERIZED. RECENTLY SEVERAL BACTERIAL SPECIES WERE IDENTIFIED THAT GREW UNDER 7 MBAR 0 C AND CO2-ANOXIC ATMOSPHERES (HENCEFORTH CALLED HYPOBAROPHILES) (SCHUERGER ET AL. 2013 SCHUERGER AND NICHOLSON 2015A 2015B). HOWEVER THESE EXPERIMENTS WERE RESTRICTED TO MOSTLY NON-SPACECRAFT MICROORGANISMS AND THUS A KEY KNOWLEDGE GAP IN PLANETARY PROTECTION IS (1) WHETHER HYPOBAROPHILE MICROORGANISMS ARE PRESENT ON ACTUAL PLANETARY SPACECRAFT SCHEDULED FOR MARS LANDINGS. OTHER PLANETARY PROTECTION KNOWLEDGE GAPS FOR MARS ROBOTIC OR CREWED MISSIONS ARE: (2) WHAT IS THE RATIO OF CULTURABLE VERSUS NONCULTURABLE HYPOBAROPHILES ON PLANETARY SPACECRAFT AND (3) WHAT ARE THE LOW-TEMPERATURE AND HIGH-SALT TOLERANCE LEVELS FOR MAINTENANCE METABOLISM AND GROWTH OF SPACECRAFT HYPOBAROPHILES IN SPECIAL REGIONS ON MARS (E.G. RSL LAVA TUBE ICE CAVES AND GROUND ICE)? ANSWERS TO THESE QUESTIONS WILL CONTRIBUTE TO MODELING THE RISKS OF FORWARD CONTAMINATION OF SPECIAL REGIONS ON MARS. THE CURRENT PROPOSAL WILL FOCUS ON CHARACTERIZING MAINTENANCE METABOLISM AND GROWTH OF SPACECRAFT HYPOBAROPHILIC BACTERIA UNDER MARTIAN CONDITIONS NEAR 7 MBAR. OUR RESULTS WILL CONTRIBUTE TO (I) CHARACTERIZING THE LIMITS OF LIFE UNDER MARTIAN CONDITIONS (II) ASSIST IN THE ENUMERATION OF TERRESTRIAL BACTERIA ON MARS ROVERS OR LANDERS PRIOR TO LAUNCH AND (III) CONTRIBUTE TO DEVELOPING PLANETARY PROTECTION PROTOCOLS FOR SANITIZING HARDWARE ON ROBOTIC AND CREWED MISSIONS TO MARS. OBJECTIVE-1: CULTURABLE SPACECRAFT HYPOBAROPHILES. WE WILL UTILIZE RECENTLY DEVELOPED PROTOCOLS TO ASSAY A RANGE OF SOILS SPACECRAFT SURFACES AND SPACECRAFT ASSEMBLY FACILITY SAMPLES FOR THE PRESENCE OF CULTURABLE HYPOBAROPHILES CAPABLE OF GROWTH AT 7 MBAR. DNA FROM HYPOBAROPHILES WILL BE SANGER SEQUENCED TO ESTABLISH THEIR TAXONOMIC AFFILIATIONS. OBJECTIVE-2: NONCULTURABLE SPACECRAFT HYPOBAROPHILES. IN ADDITION WE WILL DEVELOP A STABLE ISOTOPE PROBING (SIP) PROTOCOL COUPLED TO A NEXTGEN METAGENOMIC SEQUENCING APPROACH (ILLUMINA MISEQ) TO DETERMINE THE PRESENCE OF NONCULTURABLE HYPOBAROPHILES IN THE ABOVE SAMPLES. IT IS A MODERN PARADIGM THAT&GT;99% OF ALL ENVIRONMENTAL BACTERIA AND ARCHAEA ARE NONCULTURABLE SO WE ANTICIPATE MANY MORE NONCULTURABLE HYPOBAROPHILES TO BE DESCRIBED THAN THE CURRENT LIST OF 23 HYPOBAROPHILIC BACTERIA (SCHUERGER AND NICHOLSON 2015A 2015B). OBJECTIVE-3: GROWTH OF HYPOBAROPHILES AT LOW TEMPERATURES AND HIGH SALT CONCENTRATIONS. SPACECRAFT HYPOBAROPHILES WILL BE TESTED FOR GROWTH ON SEMI-SOLID AND LIQUID MEDIA TO DETERMINE THE MINIMUM TEMPERATURES AND MAXIMUM SALT CONCENTRATIONS REQUIRED FOR GROWTH. RESULTS WILL BE USED IN OBJECTIVE 4 AND WILL GIVE INSIGHTS INTO HOW ENVIRONMENTAL VERSATILE THE HYPOBAROPHILES MIGHT BE ON THE SURFACE OF MARS. OBJECTIVE-4: MAINTENANCE METABOLISM OF SPACECRAFT HYPOBAROPHILES. WE WILL USE NANOSIMS TO PROBE MAINTENANCE METABOLISM OF VIABLE BUT NOT DIVIDING HYPOBAROPHILIC BACTERIA BELOW THEIR RESPECTIVE LOW-TEMPERATURE AND HIGH-SALT GROWTH THRESHOLDS. NANOSIMS IS CAPABLE OF IMAGING STABLE ISOTOPES (E.G. 13C 15N) IN FIXED BACTERIAL CELLS AT 100 NM SCALES. IF THE BACTERIAL CELLS ARE METABOLICALLY ACTIVE BUT NOT CAPABLE OF CELL DIVISION THE STABLE ISOTOPES WILL BE ABSORBED AND INCORPORATED INTO CELLULAR MATERIALS. RESULTS WILL GREATLY EXPAND OUR CURRENT UNDERSTANDING OF HOW SPACECRAFT MICROORGANISMS MIGHT SURVIVE AND GROW ON THE SURFACE OF MARS IN GENERAL AND SPECIAL REGIONS IN PARTICULAR. AND THE RESULTS ARE LIKELY TO ASSIST IN CLOSING KNOWLEDGE GAPS FORESEEN IN UPCOMING HUMAN MISSIONS TO MARS.