TITLE: CHARACTERIZATION OF ISS MICROORGANISMS THAT ASSIST IN THE DECOMPOSITION OF COMPLEX ORGANIC MATTER DURING SPACEFLIGHT. KEY OBJECTIVE OF PROJECT: A BROAD ARRAY OF EXPERIMENTS ARE PERFORMED CONTINUOUSLY ON INTERNATIONAL SPACE STATION (ISS). THESE EXPERIMENTS GENERATE WASTE MATTER WHICH NEEDS TO BE DISPOSED OFF SAFELY AND COST-EFFECTIVELY E.G. DEAD PLANT MATTER GENERATED FROM THE EXPERIMENTS CARRIED OUT TO CHECK FOR THE EFFECT OF MICROGRAVITY ON THE DEVELOPMENT OF ROOTS AND CELLS ON PLANT SEEDLINGS. FILAMENTOUS FUNGI ARE USED INDUSTRIALLY AS SOURCES OF ENZYMES TO BREAK DOWN COMPLEX SUGARS FROM PLANT SOURCES. THE WANG GROUP AT THE UNIVERSITY OF SOUTHERN CALIFORNIA AND VENKATESWARAN GROUP AT JPL HAVE SENT FILAMENTOUS FUNGI ON TWO DIFFERENT SPACE FLIGHT PROJECTS (NASA MICRO-10) AND (CASIS-BRIC-NP). OUR ANALYSIS OF THESE FLIGHT SAMPLES COMPARED TO GROUND CONTROLS HAVE SHOWN DIFFERENTIAL EXPRESSION PARTICULARLY OF GENES RELATED TO CARBOHYDRATE METABOLIC PROCESSES. IN ADDITION MICROBIAL TRACKING (MT-1) PROJECT ISOLATED 202 MICROBIAL STRAINS (138 BACTERIA AND 64 FUNGI) AND ARCHIVED AT JPL WILL BE UTILIZED TO IDENTIFY THE MICROBIAL CULTURE OR CONSORTIUM TO DEGRADE PLANT ORGANIC MATTER. THIS FINDING PROVIDES FURTHER JUSTIFICATION THAT FILAMENTOUS FUNGI AND SOME UNIQUE BACTERIAL STRAINS CAN POTENTIALLY ASSIST IN THE DECOMPOSITION OF COMPLEX ORGANIC MATTER DURING SPACEFLIGHT. THE PROPOSED INVESTIGATION IS A GROUND-BASED PROJECT TO CHARACTERIZE MICROORGANISMS ISOLATED FROM THE ISS IN THE DEGRADATION OF PLANT COMPOST MATERIALS. WE WILL UTILIZE VARIOUS SIMULATED GRAVITY SYSTEMS AVAILABLE AT JPL AND NASA TO UNDERSTAND THE MECHANISM OF HOW ISS MICROORGANISMS ASSIST IN THE DECOMPOSITION OF COMPLEX ORGANIC MATTER DURING SPACEFLIGHT. THE OBJECTIVES OF THE PROJECT AND METHODS USED ARE: 1) IDENTIFY THE ISS MICROBIAL SPECIES ISOLATED DURING MT-1 PROJECT WHICH CAN DEGRADE PLANT COMPLEX ORGANIC MATTER/ UTILIZE COMPLEX SUGAR SOURCES. 2) GENETICALLY ENGINEER UP TO FIVE MT-1 MICROBIAL SPECIES (SINGLE OR SYMBIOTIC COMBINATION OF BACTERIA/FUNGI) DOWN SELECTED FROM OBJECTIVE 1 USING CRISPR/CAS9 TECHNOLOGY FOR CREATING MUTANTS THAT ARE EFFICIENT IN DEGRADING COMPLEX ORGANIC MATTER. 3) EXAMINE THE PROTEOMIC PROFILE OF FILAMENTOUS FUNGI AND BACTERIAL MUTANTS CREATED IN OBJECTIVE 2 AND WILD TYPE STRAINS USING SIMULATED MICROGRAVITY DEVICES IN COMPARISON TO GROUND CONTROL. 4) EXAMINE THE DIFFERENTIAL EXPRESSION OF CARBOHYDRATE DEGRADATION GENES AND THEIR REGULATORY GENES USING SIMULATED MICROGRAVITY DEVICES IN COMPARISON TO GROUND CONTROL OF MUTANTS AND WILD TYPE STRAINS USING WHOLE GENOME SEQUENCING AND WHOLE GENOME MRNA SEQUENCING. SIGNIFICANCE OF PROPOSED WORK: THE PROPOSED PROJECT ADDRESS SUBTOPIC PL-C EXPERIMENTS THAT DEMONSTRATE THAT SYMBIOTIC MICROBES CAN ASSIST IN THE DECOMPOSITION OF COMPLEX ORGANIC MATTER DURING SPACEFLIGHT. THE PROJECT WILL NOT ONLY PROVIDE INSIGHTS INTO THE MECHANISMS OF MICROBIAL DEGRADATION OF COMPLEX ORGANIC MATTER DURING SPACE FLIGHT BUT AID IN DEVELOPING PRACTICAL MEASURES FOR MITIGATING RISK TO HUMAN HEALTH AND ISS ENVIRONMENT THROUGH WASTE DISPOSAL. THE DATA GENERATED WILL AUGMENT THE NASA GENELAB DATABASE WHICH PROVIDES NASA WITH THE STATISTICAL CONFIDENCE TO CHARACTERIZE MICROBES ASSOCIATED WITH CLOSED HABITATION AND CREW HEALTH MAINTENANCE. THE ANALYSIS PROPOSED WILL HELP ELUCIDATE THE IMPACT OF SPACEFLIGHT STRESS ON THE PRODUCTION OF NOVEL METABOLITES AND DIRECTLY RESPONSIVE TO THE SPACE BIOLOGY DECADAL SURVEY RECOMMENDATION P1. FURTHERMORE SPECIFIC PROJECT OBJECTIVES WILL ENABLE US TO ANSWER SPACE BIOLOGY GUIDING QUESTIONS.