Effects of Simulated Space Environmental Conditions on Cleanroom Microbes

Chelsi D. Cassilly^1*Atul M. Chander²Jason A. Vaughn¹Kevin J. Kunstman³Stefan Green³Kasthuri Venkateswaran²Peter F. Bertone¹Curtis W. Bahr⁴Samantha A. Marcella⁴Heather C. Morris⁴

• ¹NASA Marshall Space Flight Center, AL 35812,, United States
• ²NASA Jet Propulsion Laboratory (JPL), La Cañada Flintridge, California, United States
• ³Rush University, Chicago, Illinois, United States
• ⁴Amentum Space Exploration Group, Huntsville, AL, United States

Microorganisms can have significant impacts on the success of NASA's missions, including the integrity of materials, the protection of extraterrestrial environments, the reliability of scientific results, and maintenance of crew health. Robust cleaning and sterilization protocols for the spacecraft and associated environments are currently in place in NASA facilities, but microbial contamination should be controlled and its impact on NASA's missions and science must be minimized. To address this, air and surfaces across cleanrooms and uncontrolled spaces at the Marshall Space Flight Center were sampled and microbial burden and diversity were analyzed. A library of 82 microbial strains was isolated, curated, characterized, and a subset (n=24) was subjected to simulated space environmental stressors, including desiccation, vacuum, proton radiation, and ultraviolet radiation. Out of these, four non-spore former species exhibiting the highest resistance to tested stressors were selected for whole genome sequencing and comparative genomic, pan-resistomics and functional analyses. Results revealed a conserved core genome among these four species, encompassing genes critical for amino acid biosynthesis, carbohydrate metabolism, and stress response mechanisms. Notably, strain Arthrobacter koreensis PPS68 displayed unique genomic features predictive of resilience to desiccation and ionizing radiation, supported by genes for oxidative stress resistance, membrane stability, and nutrient acquisition. Erwinia sp. PPS120 demonstrated notable genomic features consistent with metabolic flexibility and stress response capabilities, particularly under oxidative stress conditions. This study also established a modular protocol for evaluating microbial survival under space-relevant stressors and identified hardy nonspore-formers with potential to inform planetary protection policies. These findings emphasize the importance of understanding microbial risks in cleanroom environments to safeguard mission success and minimize the contamination of extraterrestrial environments. Beyond planetary protection, these findings are crucial for guiding microbial risk assessment and control strategies in extraterrestrial human habitats, where closed environments can impact life support systems, immune stability and infection control.