Survival of NASA-cleanroom microbial isolates under simulated space and Martian conditions

Abstract Planetary protection hinges on understanding microbial survival following sterilization procedures, the stressors of space travel, and exposure to extraterrestrial environmental conditions. This study identified 23 fungal strains isolated from NASA-spacecraft assembly cleanrooms, capable of surviving ultraviolet radiation exposure. Using experimental simulation facilities, we conducted a comprehensive assessment of microbial survivability and morphology on the most resilient spacecraft-associated microorganisms. Aspergillus calidoustus demonstrated remarkable survival under simulated Martian conditions, withstanding up to 1440 minutes of Martian solar irradiation, Mars atmospheric pressure and composition, and the presence of Martian regolith. Lethality only occurred under combined irradiation and cooling to -60°C (the mean Mars surface temperature), emphasising the synergistic effect of these conditions. Furthermore, A. calidoustus survived long-duration neutron radiation exposure (replicating ionizing space radiation doses) and dry-heat sterilization (typically used for spacecraft components). This is the first study to perform an end-to-end evaluation of eukaryotic microbial survival across conditions that occur during preparation for, travel to, and the colonization of Mars by human. The experimental facilities and chronic exposure methods utilized offer a biologically meaningful model for understanding microbial risks during long-duration space missions. The capacity for fungal conidia to survive multiple space-relevant conditions suggests their potential as forward contaminants, capable of being transported to and persisting on Mars. As current spacecraft sterilization protocols prioritize bacterial spores, this research highlights a critical gap in planetary protection strategies. In addition to offering novel insights into microbial survival and dispersal, these findings have broader implications for biocontamination within the food, pharmaceutical, and medical sectors. Introduction Sterilization by radiation or heat exposure are critical protocols to a number of high-value industries, ranging from food safety [1, 2] and pharmaceuticals [3], to space sciences [4]. However, some microorganisms exhibit remarkable adaptations, potentially allowing for survival under typically deleterious conditions. This has interesting ecological implications for many anthropological and terrestrial niches, as well as ecosystems beyond Earth. For instance, while interplanetary missions such as the active Mars rovers and the Mars Sample Return program [5] have the potential to yield some of the most exciting, contemporary scientific outputs, it must be ensured that they do not compromise the integrity of extraterrestrial ecosystems through the introduction of Earth-originating microorganisms [6]. Mars presents an array of hostile environmental factors that can significantly impact microbial survival; the thin Martian atmosphere is predominantly composed of carbon dioxide and provides only minimal protection from ultraviolet (UV) radiation, resulting in a surface-UV flux (100-280 nm) ~10 times higher than on Earth [7]. Additionally, the comparatively low surface pressure of ~6 mbar [8], the lack of atmospheric water vapour, and the average annual Martian temperature of ~ -60oC [9, 10], present a myriad of environmental challenges for any potential colonizing organism. However, various microorganisms are known to survive exposure to UV radiation [11–13], ionizing radiation [14–16], and the variable influence of desiccation and temperature extremes [17, 18]. This suggests that there is potential for microbial survival in space or on Mars. To mitigate these risks, the National Aeronautics and Space Administration (NASA) and other spacefaring agencies implement rigorous planetary protection standards, ensuring the cleanliness of extraterrestrial space and assembly cleanrooms [17, 19, 20]. Germicidal techniques are typically quantified through enumerating aerobic spore-forming bacteria. Bacillus pumilus SAFR-032 is an example that has been extensively studied in the context of the space environment [11, 12]. However, bacterial-spore detection alone may not be an accurate representation of bioburden [21]. Fungi, such as Aspergillus and Penicillium, are common contaminants in spacecraft assembly facilities and have been isolated from Mars mission assembly cleanrooms [22]. Fungal conidia also have the ability to endure extreme environmental stressors [23], including short-term exposure to limited simulated Martian conditions [19]. It is therefore critically important to consider fungal conidia as a potential concern for planetary protection, and as valuable model species for understanding the adaptions of eukaryotic life to extreme environmental conditions. This study embarks on a detailed assessment of microorganisms that could potentially serve as contaminants on Mars-bound spacecraft components. Unique fungal conidia, isolated from spacecraft assembly facilities [24], were compared against the radio-resistant microorganisms Aspergillus fumigatus (International Space Station strain [25], and B. pumilus (isolated from a spacecraft assembly facility (SAF) cleanroom [11]. Microbial survival and morphology were assessed following exposure to simulated Martian conditions (SMC), in which a state-of-the-art experimental Martian simulation facility was used to expose microbial samples with a combination of the distinct Martian UV-radiation spectrum, atmospheric composition and pressure, and cooling to the mean Martian surface temperature. When exposed to UV radiation or extremes in temperature, microbial survival is influenced by the presence of soil [26, 27]. As such, the influence of Martian regolith was also assessed during SMC experiments. Prior to reaching Mars, contaminant organisms must survive two other potentially fatal events; an extended dose of ionizing space radiation accumulated during long-duration space travel [28], and exposure to spacecraft sterilization protocols. As such, microbial viability was also assessed following chronic exposure to a Californium-252 (252Cf) source, and after performing dry heat microbial reduction (DHMR; a common spacecraft-sterilization method [20]. 252Cf is a radionuclide that provides a low-dose neutron field that closely approximates the chronic exposure patterns of galactic cosmic rays and solar particle events encountered inside spacecraft beyond low Earth orbit [28]. This research forms a comprehensive, end-to-end assessment of microbial survival during spacecraft sterilization, deep-space travel, and exposure to the Mars environment. This provides valuable insights into microbial resistance to different radiative environments and evaluates the impact of spacecraft sterilization. The survival of fungal species following high-energy UV exposure, extended ionizing-radiation doses, and sterilization protocols highlights the critical role of fungi in astrobiology and has broader implications sterilization techniques used in food science, the pharmaceutical industry and human health. This work is crucial in informing future planetary protection strategies and provides foundational knowledge into the tolerances of life to environmental extremes both on and beyond Earth.