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The question about the stability of certain biomolecules is directly connected to the life-detection missions aiming to search for past or present life beyond Earth. The extreme conditions experienced on extraterrestrial planet surface (e.g. Mars), characterized by ionizing and non-ionizing radiation, CO2-atmosphere and reactive species, may destroy the hypothetical traces of life. In this context, the study of the biomolecules behaviour after ionizing radiation exposure could provide support for the onboard instrumentation and data interpretation of the life exploration missions on other planets. Here, as a part of STARLIFE campaign, we investigated the effects of gamma rays on two classes of fungal biomolecules–nucleic acids and melanin pigments – considered as promising biosignatures to search for during the ‘in situ life-detection’ missions beyond Earth.

Biosignatures are widely sought after within the solar system and exoplanet communities. But what are we talking about, when we talk about biosignatures?The concept of a “biosignature” is widely used to suggest a link betweenanobservation and a biological cause,depending on thecontext. The term itself has, however,been defined and used in differentways in each of the differentparts of the scientific community involved in the search for past or present life on Earth and beyond. With the ongoing acceleration in the search for lifein our solar system and on exoplanets, there is a need for clarity and accuracy in the formulation and reporting of claims.This includes the discussion of false positives as well as false negatives. In this talk, resulting from the ISSI Tracers team,wediscussan inventory of questionsthat scientists and other interested parties should ask when assessing any reported detection of a “biosignature” to better understand exactly what is being claimed.This would improve clarity and accuracy in the formulation of claims and subsequent technical and public communication about some of the most profound and important questions in science and society.&nbsp

Xanthoria parietina (L.) Th. Fr. is a widely spread foliose lichen showing high tolerance against UV-radiation thanks to parietin, a secondary lichen substance. We exposed samples of X. parietina under simulated Martian conditions for 30 days to explore its survivability. The lichen’s vitality was monitored via chlorophyll a fluorescence that gives an indication for active light reaction of photosynthesis, performing in situ and after-treatment analyses. Raman spectroscopy and TEM were used to evaluate carotenoid preservation and possible variations in the photobiont’s ultrastructure respectively. Significant differences in the photo-efficiency between UV irradiated samples and dark-kept samples were observed. Fluorescence values correlated with temperature and humidity day-night cycles. The photo-efficiency recovery showed that UV irradiation caused significant effects on the photosynthetic light reaction. Raman spectroscopy showed that the carotenoid signal from UV exposed samples decreased significantly after the exposure. TEM observations confirmed that UV exposed samples were the most affected by the treatment, showing chloroplastidial disorganization in photobionts’ cells. Overall, X. parietina was able to survive the simulated Mars conditions, and for this reason it may be considered as a candidate for space long-term space exposure and evaluations of the parietin photodegradability.

The Nostoc sp. strain CCCryo 231-06 is a cyanobacterial strain capable of surviving under extreme conditions and thus is of great interest for the astrobiology community. The knowledge of its complete genome sequence would serve as a guide for further studies. However, a major concern has been placed on the effects of contamination on the quality of sequencing data without a reference genome. Here, we report the use of microfluidic technology combined with single cell sequencing and de novo assembly to minimize the contamination and recover the complete genome of the Nostoc strain CCCryo 231-06 with high quality. 100% of the whole genome was recovered with all contaminants removed and a strongly supported phylogenetic tree. The data reported can be useful for comparative genomics for phylogenetic and taxonomic studies. The method used in this work can be applied to studies that require high-quality assemblies of genomes of unknown microorganisms.

The Moon is characterized by extremely harsh conditions due to ultraviolet irradiation, wide temperature extremes, vacuum resulting from the absence of an atmosphere and high ionizing radiation. Therefore, its surface may provide a unique platform to investigate the effects of such conditions. For lunar exploration with the Lunar Gateway platform, exposure experiments in Low Earth Orbit are useful testbeds to prepare for lunar space experiments and to understand how and if potential biomarkers are influenced by extra-terrestrial conditions. During the BIOMEX (BIOlogy and Mars EXperiment) project, dried colonies of the fungus Cryomyces antarcticus grown on Lunar Regolith Analogue (LRA) were exposed to space conditions for 16 months aboard the EXPOSE-R2 payload outside the International Space Station. In this study, we investigated the stability/degradation of fungal biomarkers in LRA after exposure to (i) simulated space and (ii) real space conditions, using Raman spectroscopy, gas chromatography-mass spectrometry and DNA amplification. The results demonstrated that fungal biomarkers were detectable after 16 months of real space exposure. This work will contribute to the interpretation of data from future biological experiments in the Cislunar orbit with the Lunar Gateway platform and/or on the lunar surface, in preparation for the next step of human exploration.

Two rover missions to Mars aim to detect biomolecules as a sign of extinct or extant life with, among other instruments, Raman spectrometers. However, there are many unknowns about the stability of Raman-detectable biomolecules in the martian environment, clouding the interpretation of the results. To quantify Raman-detectable biomolecule stability, we exposed seven biomolecules for 469 days to a simulated martian environment outside the International Space Station. Ultraviolet radiation (UVR) strongly changed the Raman spectra signals, but only minor change was observed when samples were shielded from UVR. These findings provide support for Mars mission operations searching for biosignatures in the subsurface. This experiment demonstrates the detectability of biomolecules by Raman spectroscopy in Mars regolith analogs after space exposure and lays the groundwork for a consolidated space-proven database of spectroscopy biosignatures in targeted environments.

In 2022 JAXA issued an Announcement of Opportunity (AO) for receiving Hayabusa2 samples returned to Earth. We responded to the AO submitting a proposal based on using a multi-prong approach to achieve two main goals. The first goal is to address the subdued contrast of remote-sensing observations compared to measurements performed under laboratory conditions on analog materials. For this we will link the hyperspectral and imaging data collected from the spacecraft and the in-situ observations from the MASCOT lander instruments (MARA and MASCam) with laboratory-based measurements of Hayabusa2 samples using bi-directional reflectance spectroscopy under simulated asteroid surface conditions from UV to MIR/FIR achieved using three Bruker Vertex 80 V spectrometers in the Planetary Spectroscopy Laboratory. The second goal is the investigation of the mineralogy and organic matter of the samples collected by Hayabusa2, to better understanding the evolution of materials characterizing Ryugu and in general of protoplanetary disk and organic matter, investigating the aqueous alteration that took place in the parent body, and comparing the results with data collected from pristine carbonaceous chondrite analog meteorites. Spectral data will be complemented by Raman spectroscopy under simulated asteroid surface conditions, X-ray diffraction, would also allow us to define the bulk mineralogy of the samples as well as investigate the presence and nature of organic matter within the samples. In situ mineralogical and geochemical characterization will involve a pre-characterization of the sample fragments through scanning electron microscopy low voltage electron dispersive X-ray (EDX) maps, and micro IR analyses of the fragments. If allowed, a thin section of one grain will be used for electron microprobe analyses to geochemically characterize its mineralogical composition. To train our data collection and analysis methods on a realistic sample, we selected a piece of the Mukundpura meteorite, as one of the closer analogs to Ryugu’s surface (Ray et al., Planetary and Space Science, 2018, 151, 149–154). The Mukundpura chunk we selected for this study measures 3 mm in its maximum dimension, and we chose it so to have a test sample of the same size as the Hayabusa2 grain we requested in our proposal to JAXA’s AO. The test gave us confidence that we can measure with good SNR measurements in bi-directional reflectance for samples around 3 mm in size (see Figures 3, 4 below). To address our second goal the spectral data was complemented by Raman spectroscopy measured again under simulated asteroid surface conditions in our Raman Mineralogy and Biodetection Laboratory at DLR.

BIOLEX is a concept designed for in situ science on the Moon or in its orbit. As heritage of the polar and space experiment BIOMEX (Biology and Mars Experiment) on the ISS it is a more developed concept. Measurement operations on an exposure platform as well as within a micro-greenhouse device are part of this concept. The goal is to investigate the use of lunar resources as well as to analyse the stability of biomolecules as potential biosignatures serving as reference for future space exploration missions to Mars and the icy ocean moons in the outer solar system. Astrobiological exploration of the solar system is a priority research area such as emphasized by the European Astrobiology Roadmap (AstRoMap). It is focusing on several research topics, such as "Habitability" and on "Biomarkers for the detection of life". Therefore, "space platforms and laboratories", such as the EXPOSE setup installed outside the ISS, are essential to gain more knowledge on space- and planetary environments, which might be an essential basis for improvement of the robotic and human interplanetary exploration (Moon, Mars, Encedalus, Titan and Europa). In reference to these exposure platforms a new generation of hardware is needed to be installed in the lunar orbit or directly on the Moon. The BIOLEX is representing by its LOGOS (Lunar Organisms, Geo-microbiology and Organics Space Experiment) cubes such a concept combining the life detection topics with topics relevant to autonomous life supporting systems. A combination of a sample exposure device and a microhabitat for plants and microorganisms could address a tremendous number of questions from astrobiology and life sciences. The main scientific objectives for the use of BIOLEX-LOGOS cubes are: (i) in situ measurements by spectroscopy methods (such as Raman, IR, UV/VISspectroscopy) for analysis of biosignatures and their stability what is relevant for support of future life detection missions on Mars and the icy moons in the outer solar system); (ii) in situ measurements of environmental conditions (radiation, pressure/vacuum, temperature, pH, humidity) in micro-modules or compartments in reference to planned micro-habitat experiments placed on the Moon or incorporated on an exposure facility in orbit; (iii) in situ measurements of microorganisms’ activity in micro-modules / compartments in reference to planned microhabitat experiments placed on the moon or incorporated in the exposure facility in orbit. In reference to these scientific ideas the Moon is an excellent platform to operate different space experiments which will be of relevance for astrobiology, life sciences and human space missions. BIOLEX tries to fulfil a large number of scientific investigations in reference to these disciplines. The lunar environment is much harsher compared to Mars; and tests on biomolecules in this environment could provide information on their stability and therefore on the value to be used as reference for future space missions to Mars or the icy ocean moons in the outer solar system. Resources of the Moon such as the regolith or the freely available radiation on the surface could be tested by using them in a micro-greenhouse. Within this greenhouse different filters could test the optimal spectra range of the radiation.

BIOMEX (BIOlogy and Mars EXperiment) is an ESA/Roscosmos space exposure experiment housed within the exposure facility EXPOSE-R2 outside the Zvezda module on the International Space Station (ISS). The design of the multiuser facility supports—among others—the BIOMEX investigations into the stability and level of degradation of space-exposed biosignatures such as pigments, secondary metabolites, and cell surfaces in contact with a terrestrial and Mars analog mineral environment. In parallel, analysis on the viability of the investigated organisms has provided relevant data for evaluation of the habitability of Mars, for the limits of life, and for the likelihood of an interplanetary transfer of life (theory of lithopanspermia). In this project, lichens, archaea, bacteria, cyanobacteria, snow/permafrost algae, meristematic black fungi, and bryophytes from alpine and polar habitats were embedded, grown, and cultured on a mixture of martian and lunar regolith analogs or other terrestrial minerals. The organisms and regolith analogs and terrestrial mineral mixtures were then exposed to space and to simulated Mars-like conditions by way of the EXPOSE-R2 facility. In this special issue, we present the first set of data obtained in reference to our investigation into the habitability of Mars and limits of life. This project was initiated and implemented by the BIOMEX group, an international and interdisciplinary consortium of 30 institutes in 12 countries on 3 continents. Preflight tests for sample selection, results from ground-based simulation experiments, and the space experiments themselves are presented and include a complete overview of the scientific processes required for this space experiment and postflight analysis. The presented BIOMEX concept could be scaled up to future exposure experiments on the Moon and will serve as a pretest in low Earth orbit., This research was supported by the Italian Space Agency (ASI grant BIOMEX Cyano 051-R.0 to D.B., ASI grant BIOMEX MicroColonial Fungi 063-R.0 to S.O.); the German Aerospace Center (DLR-grants: Department of Infrastructure and Management, Astrobiology Laboratories through a grant DLR-FuW-Project BIOMEX (2474128)/ Department of Radiation Biology supported by the grant DLR-FuE-Projekt ISS LIFE, Programm RF-FuW, Teilprogramm 475); the German Helmholtz Association through the Helmholtz-Alliance ‘‘Planetary Evolution and Life’’; the Spanish Ministry of Economy, Industry and Competitiveness (MINECO, project SUBLIMAS ‘‘SUrvival of Bacteria and LIchens on Mars Analogs and Space,’’ ESP2015- 69810-R, 2015, to R. de la Torre, and project ‘‘CTM2015- 64728-C2-1-R’’ to L.G. Sancho); and the National Academy of Sciences of Ukraine (grant 47/2017).

BIOMEX (BIOlogy and Mars EXperiment) is an ESA/Roscosmos space exposure experiment housed within the exposure facility EXPOSE-R2 outside the Zvezda module on the International Space Station (ISS). The design of the multiuser facility supports—among others—the BIOMEX investigations into the stability and level of degradation of space-exposed biosignatures such as pigments, secondary metabolites, and cell surfaces in contact with a terrestrial and Mars analog mineral environment. In parallel, analysis on the viability of the investigated organisms has provided relevant data for evaluation of the habitability of Mars, for the limits of life, and for the likelihood of an interplanetary transfer of life (theory of lithopanspermia). In this project, lichens, archaea, bacteria, cyanobacteria, snow/permafrost algae, meristematic black fungi, and bryophytes from alpine and polar habitats were embedded, grown, and cultured on a mixture of martian and lunar regolith analogs or other terrestrial minerals. The organisms and regolith analogs and terrestrial mineral mixtures were then exposed to space and to simulated Mars-like conditions by way of the EXPOSE-R2 facility. In this special issue, we present the first set of data obtained in reference to our investigation into the habitability of Mars and limits of life. This project was initiated and implemented by the BIOMEX group, an international and interdisciplinary consortium of 30 institutes in 12 countries on 3 continents. Preflight tests for sample selection, results from ground-based simulation experiments, and the space experiments themselves are presented and include a complete overview of the scientific processes required for this space experiment and postflight analysis. The presented BIOMEX concept could be scaled up to future exposure experiments on the Moon and will serve as a pretest in low Earth orbit.