Lignocellulose is the structurally complex building block of all plants and would be the dominant waste from crop materials during spaceflight missions. NASA recognizes the complex and highly…

This $99,998.00 cooperative agreement was awarded by the National Aeronautics and Space Administration (NASA) to the Nevada System of Higher Education (NSHE) Board of Regents Sponsored Programs Office Division. The contract focuses on exploring the ability of the bacterium Kallotenue papyrolyticum and its enzymes to degrade a variety of polysaccharides, which could be useful for degrading lignocellulose waste during spaceflight missions. The contract has two main objectives: 1) to determine the functions of K. papyrolyticum glycoside hydrolase (GH) enzymes, and 2) to determine the broader substrate range and degradation products of K. papyrolyticum. This work directly supports NASA's research objective of developing spaceflight-compatible recycling of non-edible biomass. No set-aside designation was indicated for this contract.

Lignocellulose is the structurally complex building block of all plants and would be the dominant waste from crop materials during spaceflight missions. NASA recognizes the complex and highly mission dependent nature of both the quality and quantity of lignocellulose wastes. As such, technologies for degradation of diverse lignocellulose waste streams depend on diverse organisms and enzymes regardless of the downstream application (e.g., soil formation, biofuels, specialty chemicals, etc.). To address this need, we propose to explore the ability of the novel bacterium, kallotenue papyrolyticum, and its purified glycoside hydrolase (GH) enzymes to degrade a variety of polysaccharides under the broad hypothesis that this organism could be useful to degrade lignocellulose wastes during spaceflights. The proposal has two objectives. Objective 1 is to determine the functions of k. Papyrolyticum GH enzymes. Codon-optimized genes will be synthesized, expressed in e. Coli, and screened for activity against both defined and complex polymers. Enzyme kinetic parameters of promising GHS will be determined, particularly primary cellulases. The functional properties of individual GHS are foundational for understanding polysaccharide degradation and to establish the potential contributions of k. Papyrolyticum enzymes to lignocellulose depolymerization during spaceflights. Objective 2 is to determine the broader substrate range and degradation products of k. Papyrolyticum. K. Papyrolyticum will be screened for growth against a broad group of polymers. For a few polysaccharides, metabolic products will be quantified by untargeted gas chromatography/tandem mass spectrometry. Additionally, gas chromatography with flame ionization detection will be used to quantify volatile fatty acids and alcohols through time courses and to assess the effects of terminal electron acceptor availability (i.e., low or no oxygen) and simulated microgravity on production of chemicals of potential utility. Knowledge of the broader substrate range and products of polysaccharide degradation would be critical to assess biodegradation potential and direct production of biofuels or specialty chemicals. This work directly addresses the R3 research objective defined in appendix e under research project III: spaceflight-compatible recycling of non-edible biomass. Although a variety of cellulolytic microorganisms have been described, the current cache of organisms and enzymes is insufficient to degrade the diverse and recalcitrant crop wastes necessary for long-term space flight and colonization. K. Papyrolyticum is a member of the bacterial phylum chloroflexi and is therefore unrelated to other well-described cellulolytic bacteria. By focusing on a highly cellulolytic organism from a poorly explored branch on the tree of life, this project has a strong potential to add to existing cellulolytic organisms and enzymes to address NASA s spaceflight needs and also provide a template to study other cellulolytic chloroflexi.