The Mars surface/near-surface is often considered to be biocidal. Here, however, diverse lines of evidence are presented indicating that some terrestrial microbes can survive the in-situ conditions albeit in an inactive state. For the purposes of planetary protection, it is important to consider what we mean by a planetary "surface". Most microbial cells spores or other cells deposited on Mars, even those that fall on the absolute surface, will fall within pores of the regolith or become covered by its dust. They are, therefore, protected from ultra-violet radiation. Desiccating conditions and low temperatures (-40 to -70°C) can act to preserve rather than kill all microbes, potentially maintaining a cellular viability - especially for certain extremophiles - over geological timescales. Whereas salts are ubiquitous on Mars, many microbes are highly tolerant to NaCl, but salts including the chaotropes MgCl2 and perchlorates cannot access cells (or act as chaotropes) in the absence of a liquid milieu. Whereas the Mars regolith is nutrient-deplete and conditions may be acidic in places, oligotrophic conditions per se are not biocidal and many terrestrial microbes can thrive in acidic conditions (some acidophiles can proliferate below pH 0). The low temperatures of the Mars surface are not conducive to metabolic activity, but the biophysical sophistication and robust stress biology of many terrestrial microbes, and the protection afforded by Martian conditions, are likely to ensure the long-term viability of some extremophilic microbes if transported to Mars. This article is protected by copyright. All rights reserved.

Mars is known to have once sustained an environment that may have been habitable. This idea has often been combined with theories of panspermia, to suggest that any life on Mars may have had a common origin with life on Earth. These ideas typically involve meteorites produced as impact ejecta as vectors for the transport of life between planets. However, such meteorites would be subjected to the harsh conditions of space, most notably, Solar UV radiation. Some Gram-positive bacteria generate endospores, which provide protection from extreme conditions and are common models for understanding bacterial survival in space. In this study, the spore forming bacteria Bacillus subtilis and the non-spore former Staphylococcus aureus were used to investigate the survival of bacteria exposed to UVB and UVC radiation on simulated Martian regolith. Spore survival was limited upon excessive doses of UVB and UVC radiation, although this was minimised when spores were covered with lysed bacteria which provided partial protection. These results build upon previous studies and suggest that lysed bacteria from the same bacterial species might offer spores on Martian meteorites a partial shield from UV radiation and enhance their viability in panspermia models.

ABSTRACT Planetary protection hinges on understanding microbial survival following reduction 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 1,440 min 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), emphasizing the synergistic effect of these conditions. Furthermore, A. calidoustus survived long-duration neutron radiation exposure (replicating ionizing space radiation doses) and dry-heat microbial reduction technique (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 robotic exploration of Mars. 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 microbial reduction protocols prioritize bacterial spores, this research highlights a critical gap in planetary protection strategies. In addition to offering novel insights into microbial survival, these findings have broader implications for biocontamination within the food, pharmaceutical, and medical sectors. IMPORTANCE This study reveals that conidia of the fungus Aspergillus calidoustus, which was isolated from spacecraft assembly cleanrooms, can survive simulated space-relevant stressors like ultraviolet irradiation, Martian cold atmospheric pressure, regolith exposure, ionizing radiation, and specific doses of recommended dry-heat microbial reduction method for spacecraft. Such fungal resistance demonstrates that the species can survive certain space and Mars conditions previously thought to be sterilizing, highlighting a need to revise current spacecraft decontamination standards that focus mainly on bacterial spores. This study also emphasizes the need for continued microbial monitoring of spacecraft during transit from Earth to other planets, not only to achieve goals of planetary protection but also to maintain healthy closed systems for human missions. Moreover, fungal species are highlighted as biocontamination risks for food, medical, and pharmaceutical industries, which may require the need for new standards of sterilization approaches transferable to space exploration. This study reveals that conidia of the fungus Aspergillus calidoustus, which was isolated from spacecraft assembly cleanrooms, can survive simulated space-relevant stressors like ultraviolet irradiation, Martian cold atmospheric pressure, regolith exposure, ionizing radiation, and specific doses of recommended dry-heat microbial reduction method for spacecraft. Such fungal resistance demonstrates that the species can survive certain space and Mars conditions previously thought to be sterilizing, highlighting a need to revise current spacecraft decontamination standards that focus mainly on bacterial spores. This study also emphasizes the need for continued microbial monitoring of spacecraft during transit from Earth to other planets, not only to achieve goals of planetary protection but also to maintain healthy closed systems for human missions. Moreover, fungal species are hi

Discoveries of transient liquid water in the Martian polar caps and the presence of liquid lakes and subsurface oceans in icy satellites have increased the interest of scientists in the capabilities of terrestrial extremophiles to grow and remain metabolically active in these extreme environments. The principal goal of this research is to understand the metabolic capacity of the anaerobic psychrophile, Desulfotalea psychrophila, cultured at subfreezing temperatures in media containing various concentrations of sulphate minerals. In this regard, our experiments focused on the detection of D. psychrophila survival and active metabolism, employing a biochamber that can recreate Martian temperatures. Using standard bacteriological methods for determining growth, combined with molecular and enzymatic determination of sulphate reduction, we have found that D. psychrophila is capable to carry out biological processes at temperatures down to −5°C, at concentrations that range from 0.35 to 18 wt% of MgSO4, 0.1 wt% of CaSO4 and 10 to 14 wt% of FeSO4 in which the highest sulphate concentration gradually returned the biosynthetic rate to basal limits, and the lowest temperature decreased bacterial cell division. These chemical salts, whose ions are classified as chaotropes, are known to act by maintaining water molecules in liquid state at subfreezing temperatures and by altering the stability of cellular components. This ‘chaotropic effect’ could potentially benefit the microbial metabolic activity up to a concentration in which cellular viability is jeopardized. Consequently, our hypothesis is directed towards the detection of metabolic activity as an indirect measurement of the potential influence of these ions in the flexibility/functionality of biological structures that at cold temperatures are highly rigid, compact and partially/non-functional due to water freezing. Studies of this type of microorganism are critical considering the possibility of survival and colonization of psychrophilic sulphate reducers in other planets and icy satellites.

Terraforming Mars toward sustainable life-supporting ecosystems poses unprecedented challenges. Extremophilic microbes, which thrive in Earth's most extreme environments, offer promising biological strategies for initial Mars colonization providing tools for resource mobilization and atmospheric engineering. This review synthesizes experimental evidence on microbial survival Mars-simulated conditions, highlights the ecological roles of extremophilic microbial consortia in biogeochemical cycling. We further discuss cutting-edge synthetic biology approaches to enhance microbial resilience. Yet, we emphasize the need to shift focus from single-species assessments to complex, synergistically interacting microbial communities, which may hold the key to establishing self-sustaining extraterrestrial biospheres. Finally, we consider planetary protection, ethical concerns, and future research priorities to responsibly perform Mars terraforming.

panspermia ” theory, was originated from microorganisms and simple cells transported from one planet to another and able to adapt to adverse conditions in space. In this frame studying microbial communities that are adapted to live on earth in very extreme conditions, and establishing the limits to life on our own planet, could help in getting insights into the potential of other worlds to support life, and thus determining whether life as we know it could exist elsewhere in the galaxy. (Saffary et al. 2002 ). In this work we investigated the ability of some extremophilic species to survive and to resist to some stressing conditions simulating the space environment. For our study we chose four extremophilic species from different extreme environment and representing both Archaea and Bacteria domains: Sulfolobus solfataricus (Zilling et al. 1980 ) a thermo-acidophilic archeon isolated from Solfatara volcano, southern Italy; Haloterrigena hispanica (Romano et al. 2007 ) an extremely halophilic archaeon from Fuente de Piedra, southern Spain; Thermotoga neapolitana (Jannash et al. 1988 ) a thermo-anaerobic microorganism isolated from a black smoker in the bay of Naples (Italy) and finally Geobacillus thermantarcticus (Coorevits et al. 2012 ; Lama et al. 2004 ) a thermophilic microorganism isolated from geothermal soil from Mt. Melbourne, an active volcano in Antarctica. To assess their possible ability to survive in space environment, all the selected species were exposed to extreme conditions in terms of temperature, UV radiation, humidity and pressure resembling Mars conditions and that were assayed in separately or in combination (Onofri et al. 2008 ). Three different sets of conditions were assayed: storage at extreme temperatures, UV resistance (carried out separately and both in terrestrial atmosphere and pressure conditions), resistance to martian humidity and pressure (Nicholson et al. 2013 ) investigated at the same time in a Mars simulator set at terrestrial t

conditions: either to space vacuum, solar electromagnetic radiation at >110 nm and cosmic radiation (trays 1 and 3) or to simulated Martian surface conditions EXPOSE is a multi-user facility mounted outside the International Space Station (ISS) dedicated to astrobiology. EXPOSE was developed by the European Space Agency (ESA) for long-term spaceflights and was designed to allow exposure of chemical and biological samples to outer space while recording data during exposure. The results will contribute to our understanding of photobiological processes in The Biology and Mars Experiment (BIOMEX). Its objective is to measure to what extent biomolecules, such as biological pigments, cellular components, and biofilms are resistant to and able to maintain their stability under space and Mars-like conditions. The results of BIOMEX will be relevant for space-proven biosignature definition and for…