For near-future missions planed for Mars Sample Return (MSR), an international working group organized by the Committee on Space Research (COSPAR) developed the sample safety assessment framework (SSAF). For the SSAF, analytical instruments were selected by taking the practical limitations of hosting them within a facility with the highest level of biosafety precautions (biosafety level 4) and the precious nature of returned samples into account. To prepare for MSR, analytical instruments of high sensitivity need to be tested on effective Mars analogue materials. As an analogue material, we selected a rock core of basalt, a prominent rock type on the Martian surface. Two basalt samples with aqueous alteration cached in Jezero crater by the Perseverance rover are planned to be returned to Earth. Our previously published analytical procedures using destructive but spatially sensitive instruments such as nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy coupled to energy-dispersive spectroscopy revealed microbial colonization at clay-filled fractures. With an aim to test the capability of an analytical instrument listed in SSAF, we now extend that work to conventional Fourier transform infrared (FT-IR) microscopy with a spatial resolution of 10 μm. Although Fe-rich smectite called nontronite was identified after crushing some portion of the rock core sample into powder, the application of conventional FT-IR microscopy is limited to a sample thickness of <30 μm. In order to obtain IR-based spectra without destructive preparation, a new technique called optical-photothermal infrared (O-PTIR) spectroscopy with a spatial resolution of 0.5 μm was applied to a 100 μm thick section of the rock core. By O-PTIR spectroscopic analysis of the clay-filled fracture, we obtained in-situ spectra diagnostic to microbial cells, consistent with our previously published data obtained by NanoSIMS. In addition, nontronite identification was also possible by O-PTIR spectroscopic analysis. From these results, O-PTIR spectroscopy is suggested be superior to deep ultraviolet fluorescence microscopy/μ-Raman spectroscopy, particularly for smectite identification. A simultaneous acquisition of the spatial distribution of structural motifs associated with biomolecules and smectites is critical for distinguishing biological material in samples as well as characterizing an abiotic background.

Astrobiology is often defined as the study of the origin, evolution, distribution and future of life on Earth and in the Universe and thought of as a discipline. In practice though, the delineation of astrobiology-related research and corresponding groups of researchers is far from straightforward. Here, we propose to apply text-mining methods to identify researcher communities depending on thematic similarities in their published works. After fitting a latent Dirichlet allocation topic model to the complete article corpus of three flagship journals in the field – Origins of Life and Evolution of Biospheres (1968–2020), Astrobiology (2001–2020), the International Journal of Astrobiology (2002–2020) – and computing author topic profiles, researcher communities are inferred from topic similarity networks to which community detection is applied. Such semantic social networks reveal, as we call them, ‘hidden communities of interest’ that gather researchers who publish on similar topics. The evolution of these communities is also mapped through time, bringing to light the significant shifts that the discipline underwent in the past 50 years.

The establishment of the possible presence of life on Mars (past or present) is based on the study of planetary analogues, which allow in situ analysis of the environments in which living organisms adapt to often extreme conditions. Although Mars has been a candidate for hosting life, based on observations made decades ago, it is thanks to the characteristics identified in environments, mainly volcanic, that it has been possible to calibrate instruments and detail the features of the red planet. In this paper, we present a review of the main characteristics of different planetary analogues, particularly deepening the study of Antarctica, to later expose the factors studied in Deception Island that have contributed to considering it as an analogue of Mars from different perspectives. Although geological and geomorphological studies on the analogies of the island already exist, detailed analyses that present the approach of astrobiological analogues are required, thus allowing further research.

Glycine plays an essential role in a variety of biological and biochemical processes. As the smallest amino acid, glycine is especially important in studies of prebiotic chemistry and chemical evolution. The behaviour of glycine in aqueous solution under ionizing radiation fields is still not well understood. Understanding the reaction mechanism of glycine in an ionizing radiation environment may provide insights into the complex processes involved in prebiotic chemical synthesis. Such reaction conditions could provide clues about the environmental conditions that might favour the emergence of life. Numerical modelling based on reaction kinetics provides information on the feasibility of the reaction mechanisms. In this work, we developed a numerical model in Python that describes the behaviour of glycine, as prototype compound, in aqueous solution under gamma radiation. The model is based on a variety of reaction kinetics pathways that have been proposed to describe the principal reactions between glycine and the water radicals formed by ionizing radiation. The numerical results are consistent with the experiments of other researchers. We obtained similar numerical solutions from different reaction mechanisms that share the same initial reactions. The results suggest that the primary attack of water radicals on the glycine is the main factor that controls the general decay of the molar concentration of glycine and the secondary reactions do not have a strong influence, even at high doses of nearly 200 kGy. The numerical tests of the models indicate their stability with the changing initial condition of the molar concentration of glycine. This work contributes to the advancement of knowledge regarding the behaviour of glycine in aqueous solutions under ionizing radiation from a kinetic perspective. It also provides insights into their stability under conditions that are difficult to replicate in the laboratory. Finally, this work contributes to the evaluation of appropriate numerical methods for solving the system of stiff differential equations that describe the reaction mechanism of organic molecules under high radiation fields.

In ecological systems, be it a Petri dish or a galaxy, populations evolve from some initial value (say zero) up to a steady-state equilibrium, when the mean number of births and deaths per unit time are equal. This equilibrium point is a function of the birth and death rates, as well as the carrying capacity of the ecological system itself. We show that the occupation fraction versus birth-to-death rate ratio is S-shaped, saturating at the carrying capacity for large birth-to-death rate ratios and tending to zero at the other end. We argue that our astronomical observations appear inconsistent with a cosmos saturated with extraterrestrial intelligences, and thus search for extraterrestrial intelligence optimists are left presuming that the true population is somewhere along the transitional part of this S-curve. Since the birth and death rates are a-priori unbounded, we argue that this presents a fine-tuning problem. Further, we show that if the birth-to-death rate ratio is assumed to have a log-uniform prior distribution, then the probability distribution of the ecological filling fraction is bi-modal – peaking at zero and unity. Indeed, the resulting distribution is formally the classic Haldane prior, conceived to describe the prior expectation of a Bernoulli experiment, such as a technological intelligence developing (or not) on a given world. Our results formally connect the Drake equation to the birth–death formalism, the treatment of ecological carrying capacity and their connection to the Haldane perspective.

Here we present the resistance of two halophilic Archaea, Halorubrum (Hrr.) sp. AS12 and Haloarcula (Har.). sp. NS06, isolated from the brine in Lunenburg, Germany, to stress factors including desiccation, radiation and elevated perchlorate concentration. This is the first study to describe the stress resistance of halophilic Archaea isolated from the Lunenburg brine. While Hrr. sp. AS12 tolerates desiccation up to 45 days with a -log3 reduction in survival, Har. sp. NS06 displays a strong decline in viability and no detectable survival following 21 days. In contrast, Hrr. sp. AS12 was more sensitive towards X-Ray irradiation with a significant decline in viability (D10 228,2 ± 8,9 Gy) while Har. sp. NS06 showed a slight decline in survival following exposure to 1 kGy. The resistance of both strains against germicidal UV-C254nm radiation follows a similar pattern when compared to X-ray exposure with Hrr. sp. AS12 displaying more sensitivity to UV-C radiation (F10 111,6 ± 6,4 J/m2) compared to Har. sp. NS06 (F10 194,9 ± 13,7 J/m2). Exposure to He, Ar, and Fe heavy ions up to 500 Gy showed little effect on the survivability; however, the transport control of Hrr. sp. AS12 showed a strong decline (-log3 reduction) in survival. Both strains revealed increased growth in the presence of perchlorates (NaClO4 and MgClO4) with a clear preference to NaClO4 up to 5%. Our results provide a first insight into the stress resistance of these two isolates and will further develop our understanding of the parameters of life on Earth and potentially on other planets.

Based on present knowledge of atmospheric composition, a mechanism for the natural formation of vesicles in the lakes of Titan is proposed. It involves precipitation-induced spray droplets coated by a monolayer of amphiphiles. On interaction with the monolayer on the lake’s surface, bilayer membranes are being formed that encapsulate the liquid phase of the original droplet. The resulting vesicles develop thermodynamic stability by continuous compositional selection of various types of amphiphiles in a dynamic equilibrium, leading to an optimized vesicle stability. Different populations of stable vesicles may compete, initiating a long-term evolution process that could eventually result in primitive protocells. The existence of any type of vesicles on Titan would prove that early steps towards increasing order and complexity have taken place, which represent the necessary precondition for abiogenesis. A valid analytical approach could involve a laser device with combined light scattering analysis and surface enhanced Raman spectroscopy. It would allow for very sensitive detection of amphiphiles as well as for the observation of dispersed vesicles.

The study explores the vertical stratification of microbial diversity and metabolic potential in Earth’s lower atmosphere. Using 16S rRNA sequencing data spanning the planetary boundary layer to the lower stratosphere, we conducted taxonomic profiling and metabolic pathways predictions. The aim was to elucidate microbial community dynamics and their ecological roles under diverse atmospheric conditions. Methods: 51 Publicly available datasets with 3584 samples were retrieved from repositories such as Sequencing Read Archive and European Nucleotide Archive, filtered for studies employing 16S rRNA sequencing. Quality control was performed using FastQC and Trimmomatic, followed by taxonomic classification with Qiime2 and the Silva132 database. Functional pathway predictions were derived using PICRUSt2, and statistical analyses included Kruskal-Wallis tests for diversity comparisons and Mann-Whitney U tests for pathway activity. Results: Microbial diversity decreased with altitude, with the Surface Layer exhibiting the highest Shannon diversity and the significantly decreased in Low Stratosphere. Taxonomic composition shifted along the elevation gradient, with Actinobacteria and Alphaproteobacteria predominant at lower altitudes. In contrast, Bacilli and Gammaproteobacteria became more dominant at higher elevations, though they maintained a notable presence at lower sites as well. Functional analysis revealed altitude-specific adaptations, including significant upregulation of CO2 fixation pathways in the Free Troposphere Transition Layer and secondary metabolite biosynthesis in the lower stratosphere. Discussion: These findings reveal distinct microbial metabolic profiles across atmospheric layers with varying conditions such as oxygen levels, UV radiation, and nutrient availability. While these differences may represent adaptive strategies, they could also reflect source environment characteristics or selective transport processes. The conserved metabolic pathways across altitude layers suggest functional resilience despite taxonomic divergence. These results have implications for astrobiology, providing analogs for microbial life in extraterrestrial environments like Mars or Europa. In summary, this study advances our understanding of aerobiomes’ ecological roles and their potential as models for life detection in extreme environments, bridging atmospheric microbiology with astrobiological exploration.

Ionizing radiation is known to have a destructive effect on biology by causing damage to DNA, cells and the production of reactive oxygen species, among other things. While direct exposure to high-radiation dose is indeed not favorable for biological activity, ionizing radiation can and, in some cases, is known to produce a number of biologically useful products. One such mechanism is the production of biologically useful products via charged particle-induced radiolysis. Energetic charged particles interact with the surfaces of planetary objects such as Mars, Europa and Enceladus without much shielding from their rarefied atmospheres. Depending on the energy of said particles, they can penetrate several meters deep below the surface and initiate a number of chemical reactions along the way. Some of the byproducts are impossible to produce with lower-energy radiation (such as sunlight), opening up new avenues for life to utilize them. The main objective of the manuscript is to explore the concept of a Radiolytic Habitable Zone (RHZ), where the chemistry of galactic cosmic ray-induced radiolysis can be potentially utilized for metabolic activity. We first calculate the energy deposition and the electron production rate using the GEANT4 numerical model, then estimate the current production and possible chemical pathways which could be useful for supporting biological activity on Mars, Europa and Enceladus. The concept of RHZ provides a novel framework for understanding the potential for life in high-radiation environments. By combining energy deposition calculations with the energy requirements of microbial cells, we have defined the RHZ for Mars, Europa and Enceladus. These zones represent the regions where radiolysis-driven energy production is sufficient to sustain microbial metabolism. We find that bacterial cell density is highest in Enceladus, followed by Mars and Europa. We discuss the implications of these mechanisms for the habitability of such objects in the solar system and beyond.

Spaceflight missions must limit biological contamination on both the outbound and return legs to comply with planetary protection requirements. Depending on the mission profile, contamination concerns may include the potential presence of bioactive molecules as defined by NASA’s Planetary Protection policies. Thus, the present study has examined the temperature and time requirements for sufficient inactivation/degradation of an infectious, heat-stable prion protein (Sup35NM), which serves as a model bioactive molecule. Bovine serum albumin was used to establish the method parameters and feasibility. Differential scanning calorimetry, Fourier transform infrared spectroscopy, analytical reversed-phase high-performance liquid chromatography, and mass spectrometry were utilized to analyze heat-treated samples, with non-treated samples serving as controls. Heat treatment at 400°C for 5 seconds was found to result in substantial decomposition of Sup35NM. In addition to the disruption of the protein backbone amide bonds, the side chain residues were also compromised. Fragments of molecular weight <4600 were observed by mass spectrometry but the impact of treatment on both the backbone and side chains of Sup35NM suggested that these fragments would not self-associate to create potentially pathogenic entities. The present methodology provided insight into the protein degradation process and can be applied to a variety of treatment strategies (e.g., any form of sterilization or inactivation) to ensure a lack of protein-based contamination of isolated extraterrestrial specimens.

Astrobiology is a scientific field that is very interdisciplinary and developing very fast, with many new discoveries generating a high level of attention in both the scientific community and the public. A central goal of astrobiology is to discover life beyond Earth which is, with our current instrumentation and knowledge, arguably within our reach. However, knowledge exchange crossing disciplinary boundaries is becoming increasingly challenging due to different usage of nomenclature and scientific controversies often limited to subdisciplines. There have been some efforts to compile organized databases of terms, concepts and other relevant material within some of the subfields contributing to astrobiology, for example through manually curated online portals designed to benefit students, teachers and practitioners of astrobiology-related research. However, the developments within the subfields and the potentially premature communication of research findings are too fast for objective research portals to remain reliable and up-to-date enough to enable well-informed scientific discussions. We suggest here a novel strategy for developing an online tracers portal as a self-maintaining and self-updating information platform, that would allow not only for a relatively unbiased selection of research results, but also provide fast access to latest scientific discoveries together with potential controversies, such that users of the tracers portal can form their own opinion on all available data rather than obtaining an already filtered and potentially biased selection of information.

We have initiated a large project on identifying the requirements for developing a realistic and ground-up approach to simulating the formation of terrestrial planets in our solar system. As the first phase of this project, we present here the criteria that any model of planetesimal growth needs to fulfill in order to be self-consistent and produce reliable results. We demonstrate how these criteria emerge by revisiting runaway growth and carrying out a thorough analysis of its results. As our goal is to identify the pathway to a realistic model, we focus analysis on simulations where at the beginning, planetesimals are not artificially enlarged. We show how using uninflated planetesimals, as the first requirement for a realistic model, will result in a set of criteria naturally emerging from the evolution of the system. For instance, the growth times in simulations with uninflated planetesimals become comparable to the time of giant planet formation implying that any realistic simulation of planetesimal growth, in addition to using real-size planetesimals, needs to include the perturbation of the growing giant planets as well. Our analysis also points to a strong connection between the initial distribution of planetesimals and the final outcome. For instance, due to their natural expansion, initially isolated distributions, or a collection of initially isolated distributions, such as rings of planetesimals, do not produce reliable results. In a self-consistent and realistic model, where the initial conditions are supported by basic principles and do not include simplifying, ad hoc assumptions, the entire disk of planetesimals has to be simulated at once. We present the results of our analyses and discuss their implied criteria.

Understanding microbial adaptations to the extreme conditions of space is crucial for both astronaut health and the integrity of spacecraft materials. This study comparatively analyses the cosmic radiation resistance and growth responses to simulated microgravity (SMG) of a wild-type strain and an International Space Station (ISS) isolate of Penicillium rubens. Resistance to helium- and iron-ion radiation was determined, alongside growth under SMG using clinorotation. The results revealed that the ISS isolate exhibited higher resistance to both helium- and iron-ion radiation than the wild-type strain, suggesting adaptive mechanisms that enhance survival in space environments. Additionally, while the ISS isolate demonstrated significantly increased growth in SMG compared to normal gravity conditions, the wild-type strain showed no difference between the two conditions. These findings indicate that prolonged exposure to the space environment may select for traits that enhance resistance to cosmic radiation and alter growth dynamics under microgravity. Such adaptations could have implications for microbial monitoring in space habitats, planetary protection policies, and potential biotechnological applications in space. Further investigations into the genetic and metabolic differences between both strains may provide deeper insights into fungal adaptation to space environments.

Two desert cyanobacterial strains, Chroococcidiopsis sp. CCMEE 010 and CCMEE 130, capable far-red light photoacclimation (FaRLiP), were investigated for the stability of biosignatures after six years of desiccation. Biosignature detectability was demonstrated by confocal laser scanning microscopy and Raman spectroscopy thus highlighting that these two FaRLiP cyanobacteria are a novel reservoir of an array of pigments, encompassing canonical chlorophyll a, far-red shifted chlorophylls, phycobilins and carotenoids. The recorded signals were comparable to those of dried cells of Chroococcidiopsis sp. CCMEE 029, CCMEE 057 and CCMEE 064, not capable of FaRLiP acclimation and previously reported for biosignature stability and survivability after exposure to space and Mars-like conditions during the BIOMEX (BIOlogy and Mars EXperiment) and BOSS (Biofilm Organisms Surfing Space) low Earth orbit missions. Since infrared-light driven photosynthesis has implications for the habitability of Mars as well as exoplanets, the stability of far-red shifted chlorophylls in dried Chroococcidiopsis is a prerequisite for future experimentations under simulated planetary conditions in the laboratory or directly into space. It is anticipated that post-flight investigations of FaRLiP cyanobacteria as part of the BioSigN (Bio-Signatures and habitable Niches) space mission will contribute to gather novel insights into biosignature degradation/stability and thus prepare future planetary exploration missions to Mars. In addition, the scored viability of strains CCMEE 010 and CCMEE 130 after prolonged desiccation is relevant to investigate life endurance under deep space conditions, as planned by the BioMoon mission that aims to expose dried and rehydrated extremophiles on the Moon surface after exposure to deep space.

For shallow ponds to be contenders for the venue of the emergence of life on Earth, they would have had to provide sufficient protection from ultraviolet (UV) radiation to allow for the preservation of organic molecules. Shallow ponds of a variety of compositions are proposed for early Earth, many of which may have provided ample shielding effects by attenuating UV light via absorption by (in)organic ions. Here, we present an experimental setup designed to simulate an irradiated water column to investigate the preservation/degradation of organic molecules and by proxy the attenuation of UV radiation in ponds of diverse compositions. In this setup, we dissolved glycine in ultrapure water, ferrocyanide and carbonate pond simulants and irradiated for several days. Our findings indicate that glycine’s photochemical degradation under UV irradiation is minimal in the carbonate pond, though significant in the ferrocyanide pond and in ultrapure water, where it breaks down into diverse products including formamide, glycinamide, glycinmethylester and acetaldehyde. Though ferrocyanide is a potent UV absorber, our experiments show ferrocyanide ponds to be transiently UV-shielding environments due to the removal of ferrocyanide by UV-induced precipitation of goethite and pyrite mineral assemblages and subsequent photodegradation of glycine in the cleared water column. Our results further suggest that hypersaline, carbonate ponds may present stable environments for prebiotic chemistry while providing ample UV attenuation, ultimately protecting the integrity of organic molecules. This work contributes to understanding the interplay between UV irradiation and (in)organic compounds in ponds and the suitability of those ponds for the onset of prebiotic chemistry on Earth, Mars and other celestial bodies.

If life ever existed on Mars, it may have developed survival strategies similar to those adopted by extremophiles living in terrestrial Martian analogs, such as the cryptoendolithic communities found in the rocky substrates of the McMurdo Dry Valleys or other ice-free areas of continental Antarctica. Nearly thirty years of research on these super-adapted organisms laid the foundation for the CRYPTOMARS project, which aims to disclose the genomic and phenotypic features allowing these microbial communities to withstand specific physico-chemical stresses that may be encountered on the Red Planet. This information will provide tools to outline, in terms of diversity and competences, a putative microbial community able to survive, adapt or even perpetuate under early or present Martian conditions. The project and the background information are here presented.

Due to the nascency of synthetically derived biological systems, there is a need to develop protocols for safety and security management. These protocols can be adapted from existing safety and security protocols (e.g., Biosafety Level Classification of biological agents) as well as NASA’s and ESA’s planetary protection guidelines. Currently, NASA is preparing for its first sample return mission from Mars including determining how to manage the types of hazards that may be returned to Earth. Synthetic biology can look to risk management practices from related disciplines, and NASA can look to its established protocols from lunar exploration as it strives to minimize Mars sample return bio-risk. Notably, the biosafety concerns of synthetic cell research are very similar to those of planetary back-contamination from extraterrestrial samples. Thus, the measures taken to limit planetary back-contamination can serve to help develop biosafety protocols for synthetic cell research. We summarize existing tools used in planetary protection that can be repurposed to establish protocols for synthetic cell safety and security.

Although the limits of life under individual extremes have been extensively studied, systematic experiments to quantify how combined extremes set the limits to life are lacking. We investigated the combined effects of extremes in temperature, salinity (NaCl) and pH on the growth limits of the marine bacterium Halomonas hydrothermalis, to test the hypothesis that limits to growth under combinations of the extremes establish a more restricted niche than the individual extremes. We show that the combination of supra-optimal temperature, pH and NaCl act synergistically in defining the limits of growth under multiple extremes. Although at optimal growth temperatures (30°C) maximum growth was achieved at pH 7, the maximum temperature limit of 43°C was achieved at pH 8. Under these conditions, the maximum NaCl concentration limit was 6.58% (wt/vol). Decreasing the temperature to 42 and 41/40°C increased the salinity limit to 7.01 % and 8.24 %, respectively. These data show that multiple extremes restrict the limits to growth of this organism to a greater extent than individual extremes and show how natural environments with extremes of temperature, pH and salinity could have restricted microbial diversity, or be uninhabitable, even when each individual extreme lies within the bounds of known microbial growth. These data imply that ‘maps’ of the limits to the biosphere based on laboratory-derived individual extremes may over-exaggerate growth limits in natural environments, which are rarely subject to single extremes, highlighting the need for multi-parameter analyses.

The search for biosignatures of past microbial life has promoted the interest in halophilic archaea trapped inside fluid inclusions of salt crystals. These hypersaline environments are promising targets for the preservation of microbial cell envelope biomolecules. In this study, we focused on the preservation of bacterioruberin, a carotenoid pigment found in the cell envelope of Halobacterium salinarum, within fluid inclusions of salt crystals mimicking early Mars environments and modern Earth. Halite (NaCl) and sylvite (KCl) crystals were subjected to Mars-like proton irradiation, and the preservation of carotenoids was assessed using in situ and ex situ Raman spectroscopy. Our findings demonstrate that Raman spectroscopy efficiently detected carotenoids within fluid inclusions in non-irradiated crystals. However, post-irradiation analyses posed great challenges due to fluorescence induced by the formation of colour centres in the crystal lattice, which suppressed the carotenoid signal. Cleavage of irradiated crystals revealed preserved carotenoid pigments beyond the radiation penetration depth, suggesting potential preservation of biomolecules in deeper inclusions within larger crystals. Furthermore, in some cases, carotenoids were detected even within fluorescent zones, suggesting extensive preservation. This study underscores the potential of Raman spectroscopy for the detection of carotenoids as biosignatures in planetary exploration contexts, particularly as a preliminary screening tool. However, it also highlights the need for optimized protocols to overcome fluorescence-related limitations. These findings contribute to the methodologies for detecting and interpreting biosignatures in salt deposits, advancing the search for possible traces of past microbial life beyond Earth.

Spacecraft assembly facilities (SAFs) house clean rooms where interplanetary spacecraft are built, thereby reducing the bioburden on spacecraft to protect planetary environments from terrestrial microbes that may interfere with the search for life or disturb potential native ecosystems. The most plausible environments for living systems on celestial bodies involve brines with depressed freezing points. Here, we specifically measure the abundance of salinotolerant microbes on SAF surfaces. Most probable number analyses performed with salty liquid media were applied to washes of SAF floor wipes. Microbial abundance was measured using Salt Plains medium at low salt or supplemented with (all w/v) 10% NaCl (1.7 M; aw = 0.92), 50% MgSO4 (2.0 M as epsomite; aw = 0.94), 5% NaClO3 (0.5 M; aw = 0.98), or 5% NaClO4 (0.4 M; aw = 0.98). The abundance of salinotolerant microbes was generally 1 to 10% (102 to 104 cells m−2) of the total population of microbes observed in low-salt medium (105 cells m−2). Microbes were isolated by repetitive streak-plating of positive enrichment cultures and then characterized. All of the 38 isolates were Gram-positive bacteria, mainly spore-forming Bacillaceae, with some Staphylococcus. The isolate collection showed strong tolerance to high concentrations of NaCl (to 30%), MgSO4 (to 50%) and sucrose (to 70%). There also was substantial tolerance to pH (5 to 10) and temperature (4 to 60 °C). Taken together, these SAF isolates are polyextremophiles that are in substantial abundance in the clean rooms where spacecraft are assembled.