Andreas Elsaesser

Prokaryotes, particularly those in extreme environments, are capable of diverse metabolic states resulting in altered cell envelope structure and function. However, these changes are difficult to assess as standard fluorescent probes are often incompatible with extreme conditions and/or extremophile cell physiology. Halophilic archaea present the challenge of near-saturated intra-/extra-cellular salts, high membrane potential, and extended survival in altered metabolic states including entrapment within salt crystal fluid inclusions. We evaluated the compatibility of six fluorescent markers of cell envelope stability and activity with two model species, Halobacterium salinarum and Haloferax volcanii . Redox activity markers alamarBlue and pure resazurin solutions, membrane potential probes MitoTracker Orange-CMTMRos and Rhodamine 123, and SYTO 9 and propidium iodide (LIVE/DEAD kit) to assess cell membrane integrity were evaluated for use in bulk (microplate reader) and cell-specific (microscopy) applications. Limitations of each probe were identified, clarifying the utilization of each based on cell physiology, growth phase, medium composition, and probe exposure time including extended timescales needed to simulate the environmental conditions of haloarchaea. Of particular note, propidium iodide behavior was unreliable leading to double-labeling of cells and false interpretation of cells as dead. These data provide important insights into the study of prokaryotes in non-standard conditions.

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.

Space-based platforms currently represent the most accurate means to experimentally assess the influence of the space environment on biological systems. However, performing such experiments remains technically challenging and requires highly specialized instrumentation. This study describes the current development and hardware qualification of ExocubeBio, a miniaturized experimental platform for in-situ biological space exposure. This experiment is scheduled for installation on the exterior of the International Space Station in 2027, as part of Exobio, the European Space Agency’s new generation exobiology exposure facility. ExocubeBio will expose live microbial samples to the low Earth orbit environment, and combine autonomous in-situ optical density and fluorescence measurements, with the capacity to return preserved samples to Earth. Achieving these experimental goals requires a specialized, robust and reliable hardware system. The ExocubeBio hardware testing described here includes assessment of material biocompatibility and durability, functional validation of the miniaturized fluidic system, and optimization of the integrated optical subsystem for optical density and fluorescence measurements. These results demonstrate that the ExocubeBio experimental hardware components can each execute their core functional and operational requirements; subsystems allow for sample exposure, in-situ measurements of microbial cultures, and the chemical preservation of samples for post-flight analysis. As ExocubeBio transitions from hardware development to mission readiness, the results presented here validate the overall design and engineering approaches utilized. By combining the strengths of in-situ monitoring and sample return, ExocubeBio represents a significant advancement in space-based experimentation, and will provide new insights into microbial responses to the space environment.

The formation and alteration of organic molecules within the Solar System and beyond is closely related to the life cycle of ice in space. Cold molecular clouds in the interstellar medium (ISM) are regions, where volatile molecules condense onto submicron-sized dust grains. Throughout their lifetime, these interstellar ice particles are exposed to energetic photons, charged particles and thermal processing, which catalyze the formation of increasingly complex organic compounds and induce the desorption of volatile species. The accretion of these compounds into comets and asteroids would allow their delivery on planetary surfaces, making them potential precursors of the building blocks of life. Alongside comets and asteroids, complex organics are also present on the icy moons of giant planets which are targets in both NASA’s and ESA’s space exploration programs. In this paper, we present a new experimental setup, built to study the formation and alteration of complex organic molecules of astrobiological-relevance in cryogenic environments. Possible samples include inorganic and organic molecules, in contact with various ice mixtures (e.g., H₂O, NH₃, CO, CO₂, CH₃OH) which can be exposed to UV photons, electron bombardment and undergo thermal processing. The setup allows continuous monitoring by Fourier transform-infrared (FT-IR) transmission spectroscopy during the ice formation, irradiation-, and warm-up. We present FT-IR measurements of pure H₂O, pure NH₃ and pure CH₃OH ice samples deposited at 15 K and we show the main spectral changes induced by heating and electron bombardment. Furthermore, we show the formation of new molecular species within NH₃:CH₃OH and NH₃:CH₃OH:H₂O ice mixture under electron bombardment, and outline an experimental design used to investigate the stability of organic molecules under simulated conditions on icy moons. With our setup we envision to provide spectral information on astrobiologically-relevant molecules, such as organic species fundamental for living organisms and potential biosignatures, in contact with ices of relevance for astrophysics and planetary science. Graphical Abstract

Hypersaline environments, including brines and brine inclusions of evaporite crystals, are currently of great interest due to their unique preservation properties for the search for terrestrial and potentially extraterrestrial biosignatures of ancient life. However, much is still unclear about the specific effects that dictate the preservation properties of brines. Here we present the first insights into the preservation of cell envelope fragments in brines, characterizing the relative contributions of brine composition, UV photochemistry, and cellular macromolecules on biosignature preservation. Cell envelopes from the model halophile Halobacterium salinarum were used to simulate dead microbial cellular remains in hypersaline environments based on life as we currently know it. Using different Early Earth and Mars analogue brines, we show that acidic and NaCl-dominated brine compositions are more predisposed to preserving complex biosignatures from UV degradation, but that the composition of the biological material also influences this preservation. Furthermore, a combinatory effect between chaotropicity and photochemistry occurs, with the relative importance of each being brine-specific. These results provide an experimental framework for biosignature detection in hypersaline environments, emphasizing the need for laboratory simulations to evaluate preservation properties of each potential brine environment, on Earth and elsewhere in the solar system.

The Photochemistry on the Space Station (PSS) experiment was part of the European Space Agency’s EXPOSE-R2 mission and was conducted on the International Space Station from 2014 to 2016. The PSS experiment investigated the properties of montmorillonite clay as a protective shield against degradation of organic compounds that were exposed to elevated levels of ultraviolet (UV) radiation in space. Additionally, we examined the potential for montmorillonite to catalyze UV-induced breakdown of the amino acid alanine and its potential to trap the resulting photochemical byproducts within its interlayers. We tested pure alanine thin films, alanine thin films protected from direct UV exposure by a thin cover layer of montmorillonite, and an intimate combination of the two substances forming an organoclay. The samples were exposed to space conditions for 15.5 months and then returned to Earth for detailed analysis. Concurrent ground-control experiments subjected identical samples to simulated solar light irradiation. Fourier-transform infrared (FTIR) spectroscopy quantified molecular changes by comparing spectra obtained before and after exposure for both the space and ground-control samples. To more deeply understand the photochemical processes influencing the stability of irradiated alanine molecules, we performed an additional experiment using time-resolved FTIR spectroscopy for a second set of ground samples exposed to simulated solar light. Our collective experiments reveal that montmorillonite clay exhibits a dual, configuration-dependent effect on the stability of alanine: while a thin cover layer of the clay provides UV shielding that slows degradation, an intimate mixture of clay and amino acid hastens the photochemical decomposition of alanine by promoting certain chemical reactions. This observation is important to understand the preservation of amino acids in specific extraterrestrial environments, such as Mars: cover mineral layer depths of several millimeters are required to effectively shield organics from the harmful effects of UV radiation. We also explored the role of carbon dioxide (CO ₂ ), a byproduct of alanine photolysis, as a tracer of the amino acid. CO ₂ can be trapped within clay interlayers, particularly in clays with small interlayer ions such as sodium. Our studies emphasize the multifaceted interactions between montmorillonite clay and alanine under nonterrestrial conditions; thus, they contribute valuable insights to broader astrobiological research questions.

As humans advance their presence in space and seek to improve the quality of life on Earth, a variety of science questions in support of these two objectives can be answered using the Moon. In this paper, we present a concept for an integrated mission focused on answering fundamental and applied biological questions on the Moon: BioMoon. The mission was designed to investigate the effects of the lunar radiation, gravity, and regolith on biological systems ranging from biomolecules to systems with complex trophic interactions, spanning a range of model organisms. Using common analytical systems and data processing, BioMoon represents a systems-level integrated life sciences mission. It would provide fundamental insights into biological responses to the lunar environment, as well as applied knowledge for In-Situ Resource Utilisation (ISRU), closed-loop life support system development, planetary protection and human health care. The mission was conceived to test biotechnology and sensor technology for lunar and terrestrial application and provide education and outreach opportunities. Although BioMoon was considered in the context of the European Space Agency’s Argonaut (European Large Logistics Lander) concept, the mission design provides a template for any integrated life sciences experimental suite on the Moon and other celestial bodies, implemented either robotically or by human explorers.