The spread of antibiotic resistance is becoming a serious global health concern. Numerous studies have been done to investigate the dynamics of antibiotic resistance genes (ARGs) in both indoor and outdoor environments. Nonetheless, few studies are available about the dynamics of the antibiotic resistome (total content of ARGs in the microbial cultures or communities) under stress in outer space environments. In this study, we aimed to experimentally investigate the dynamics of ARGs and metal resistance genes (MRGs) in Kombucha Mutualistic Community (KMC) samples exposed to Mars-like conditions simulated during the BIOMEX experiment outside the International Space Station with analysis of the metagenomics data previously produced. Thus, we compared them with those of the respective non-exposed KMC samples. The antibiotic resistome responded to the Mars-like conditions by enriching its diversity with ARGs after exposure, which were not found in non-exposed samples (i.e., tet and van genes against tetracycline and vancomycin, respectively). Furthermore, ARGs and MRGs were correlated; therefore, their co-selection could be assumed as a mechanism for maintaining antibiotic resistance in Mars-like environments. Overall, these results highlight the high plasticity of the antibiotic resistome in response to extraterrestrial conditions and in the absence of anthropogenic stresses.
A kombucha multimicrobial culture (KMC) was exposed to simulated Mars-like conditions in low Earth orbit (LEO). The study was part of the BIOlogy and Mars EXperiment (BIOMEX), which was accommodated in the European Space Agency’s EXPOSE-R2 facility, outside the International Space Station. The aim of the study was to investigate the capability of a KMC microecosystem to survive simulated Mars-like conditions in LEO. During the 18-month exposure period, desiccated KMC samples, represented by living cellulose-based films, were subjected to simulated anoxic Mars-like conditions and ultraviolet (UV) radiation, as prevalent at the surface of present-day Mars. Postexposure analysis demonstrated that growth of both the bacterial and yeast members of the KMC community was observed after 60 days of incubation; whereas growth was detected after 2 days in the initial KMC. The KMC that was exposed to extraterrestrial UV radiation showed degradation of DNA, alteration in the composition and structure of the cellular membranes, and an inhibition of cellulose synthesis. In the ‘‘space dark control’’ (exposed to LEO conditions without the UV radiation), the diversity of the microorganisms that survived in the biofilm was reduced compared with the ground-based controls. This was accompanied by structural dissimilarities in the extracellular membrane vesicles. After a series of subculturing, the revived communities restored partially their structure and associated activities.
As part of the ESA space experiment BIOMEX (Biology and Mars Experiment) the lichen Buellia frigida has been exposed to space and simulated Mars analogue conditions on the expose facility EXPOSE-R2 placed outside the Russian Zvezda module on the International Space Station (ISS) for 1.5 years. Randomly Amplified Polymorphic DNA (RAPD) technique has been carried out to study the effect of space conditions on the DNA integrity as well as to assess DNA damage. The RAPD profiles of the space exposed lichen samples demonstrate conspicuous changes compared to the control profiles. For the survival of cells and entire organisms the DNA integrity is an essential prerequisite. The results of the study presented indicate a minor resistance potential of the lichen Buellia frigida towards Low Earth Orbit and Mars analogue conditions effecting the survival potential and the resistance of the symbiotic organism.
This volume on astrobiology of the Springer Briefs in Life Sciences book series addresses the three fundamental questions on origin, evolution, distribution and future of life in the universe: how does life begin and evolve? Is there life beyond Earth and, if so, how can we detect it? What is the future of life on Earth and in the universe? The book provides insights into astrobiological experiments that are being performed on the International Space Station, ISS, and discusses their findings. This extremely exciting volume on astrobiology is intended for scientists of various research fields and for laypersons interested in space research and in the fundamental issues of the universe and life.
Experiments in space and in simulated planetary conditions are needed to support and to prepare future planetary exploration missions to Mars, Enceladus, Europa and/or Titan. As part of several ESA space missions, samples of extremophile lichens have been exposed to extraterrestrial conditions, i.e. space-and Mars like parameters, during short (FOTON satellite M-3) and long periods (Why lichens? Because they are extremophile organisms high tolerant to environmental stress: UV-radiation, desiccation, extreme temperatures, with the next characteristics: 1) simple ecosystem: autotroph and heterotroph 2) necessary for the re-definition of limits of life 4) selection hábitats/ planetary analog áreas: Canary Islands, high mountains, deserts, Antarctica, etc.) The lichen species exposed to space and Mars-like conditions: Rhizocarpon geographicum, Xanthoria elegans, Circinaria gyrosa, Stereocaulon vesubianum, and Xanthoparmelia hueana The results showed that X. elegans, C. gyrosa and X. hueana showed the highest survival to the extraterrestrial environment, demonstrated by physiological (PSII activity, vitality by Confocal Laser Scanning Microscopy CLSM, Transmission Electron Microscopy TEM) and ultrastructural studies. Additionally, the studies on the stability of biomolecules, like the whewellite  we have identified with RAMAN spectroscopy, will be a relevant aspect helping to answer the question of what are suitable and promising biohints [5, 6] to focus on in the search of life. pre-flight post-flight flight control spare control Fv/Fm 0 200 400 600 800 n = 4 n = 8 pre-flight post-flight flight control spare control Fv/Fm 0 200 400 600 800 pre-flight post-fl ight flight control gr ound control Fv/Fm 0 200 400 600 800 > 110 nm > 200 nm > 290 nm > 400 nm Aspicilia fru ticu lo sa 72 h n = 4 n = 8 Space UV+Vacuum Mars-UV + Atm  Sancho, L.G., and team of LICHENS (2007) Lichens survive in space: results from the 2005 LICHENS experiment. Astrobiology 7:443-454.  de la Torre and team of LITHOPANSPERMIA (2010) Survival of lichens and bacteria exposed to outer space conditions-results of the Lithopanspermia experiments.
The discovery of life on other planets and moons in our solar system is one of the most important challenges of this era. The second ExoMars mission will look for traces of extant or extinct life on Mars. The instruments on board the rover will be able to reach samples with eventual biomarkers until 2 m of depth under the planet’s surface. This exploration capacity offers the best chance to detect biomarkers which would be mainly preserved compared to samples on the surface which are directly exposed to harmful environmental conditions. Starting with the studies of the endolithic meristematic black fungus Cryomyces antarcticus, which has proved its high resistance under extreme conditions, we analyzed the stability and the resistance of fungal biomarkers after exposure to simulated space and Mars-like conditions, with Raman and Gas Chromatography–Mass Spectrometry, two of the scientific payload instruments on board the rover.
This volume on astrobiology of the Springer Briefs in Life Sciences book series addresses the three fundamental questions on origin, evolution, distribution and future of life in the universe: how does life begin and evolve? Is there life beyond Earth and, if so, how can we detect it? What is the future of life on Earth and in the universe? The book provides insights into astrobiological experiments that are being performed on the International Space Station, ISS, and discusses their findings.
The future search-for-life missions to Mars - ESA/Roscosmos’s ExoMars2020 and NASA’s Mars2020 rovers - will carry Raman spectrometers for in situ analysis of extraterrestrial material for the first time1,2. The question remains whether signs of extinct or extant life could be detected by this method. From our terrestrial examples, carotenoids (e.g. serving in cyanobacteria as accessory and photoprotective pigments) have been extensively used as biosignature models due to their stability and easy identification by Raman spectroscopy with a 532nm excitation wavelength3. Evaluating the detection limit of pigments under simulated extraterrestrial conditions is beneficial for the success of future life-detection missions. Ionizing radiation can be considered the most deleterious factor for the long term preservation of potential biomarkers on Mars4. Here, we report on the preservation potential of Raman signatures in the Antarctic cyanobacterium Nostoc sp. strain CCCryo 231–06 after high doses of gamma irradiation performed in the frame of the STARLIFE project5. The carotenoids' signals usually dominate the Raman spectra at 532nm excitation wavelength due to resonance effects. But comparing their distribution and quantifying their preservation is still problematic in natural samples. To standardize the analyses, we successfully applied Raman mapping and signal-to-noise ratios (SNR) masks to quantify the effects of irradiation. The typical in vivo Raman signatures of carotenoids could be detected even after exposure to up to 56 kGy with significant deterioration in terms of signal coverage and SNR. However, for colonies embedded in two different Martian mineral analogues (phyllosilicatic and sulfatic Mars regolith simulants), the carotenoids' signatures remained detectable even after the highest dose of γ-rays (117kGy) tested in this study, with no significant effect on signal coverage or SNRs. Carotenoids proved again their scientific value as model biosignatures for future life detection missions on Mars. Data gathered during these ground-based irradiation experiments contribute to interpret results from space experiments (such as BIOMEX6) and will guide our search for life on Mars and other bodies of interest.
Mars and the Jovian and Saturnian moons (Europa and Enceladus) are the next targets to search for life in our Solar System. New life detection instruments are indeed ready to be sent to Mars in 2020 (onboard ESA/Roscomos’s ExoMars2020 and NASA’s Mars2020 rovers) and possibly further. Among them, spectroscopy methods such as Raman or infrared are promising techniques that can give insights on both the mineralogical context and the identification of biosignatures. However, to support and interpret spectroscopic data correctly, as well as to guide future life detection missions, a better understanding of possibly habitable environments and potentially detectable biosignatures is of paramount importance. During the last years extensive field and laboratory investigations focused on demonstrating the capabilities of such technologies to characterize both mineral and biological samples of relevance to Mars but very few assessed potential biosignatures degradation under Mars-like or space-like conditions. To this end we are using samples from ground-based and space exposure experiments, the STARLIFE  and the BIOMEX  projects, to characterize their Raman and IR signatures after space and Mars relevant stresses. BIOMEX was part of the EXPOSE-R2 mission of the European Space Agency, which allowed a 15-month exposure on the outer side of the International Space Station and STARLIFE is an international campaign to study the role of galactic cosmic radiation in astrobiological systems. A wide range of extremophilic organisms such as cyanobacteria, permafrost green-algae, iron bacteria or methanogens and selected biomolecules exposed under these conditions will help us to define targets for future missions to Mars (and other bodies) carrying Raman, IR or LIBS spectrometers and give further clues about the potential habitability of Mars. We report, as an example, on the preservation potential of cyanobacterial photoprotective pigments (carotenoids) in the Antarctic cyanobacterium Nostoc cf. punctiforme strain CCCryo 231-06 after high doses of gamma irradiation and after space exposure .  R. Moeller, M. Raguse, S. Leuko, T. Berger, C.E. Hellweg, A. Fujimori, R. Okayasu, and G. Horneck, Astrobiology, 17, 101–109 (2017).  J.-P. de Vera, M. Alawi, T. Backhaus, M. Baqué, D. Billi, U. Böttger, T. Berger, M. Bohmeier, C. Cockell, R. Demets, R. de la Torre Noetzel, H. Edwards, A. Elsaesser, C. Fagliarone, A. Fiedler, B. Foing, F. Foucher, J. Fritz, F. Hanke, et al., Astrobiology, 19, 145–157 (2019).  M. Baqué, F. Hanke, U. Böttger, T. Leya, R. Moeller, and J.-P. de Vera, Journal of Raman Spectroscopy, 49, 1617–1627 (2018).
The future robotic exploration missions to Mars—European Space Agency/Roscosmos's ExoMars2020 and National Aeronautics and Space Administration's Mars2020 rovers—will search for signs of extant or extinct life using, among other instruments, Raman spectrometers for the first time. The question remains whether organic biosignatures—such as pigments and cellular components—may be detected by this method. Evaluating their detection limit under simulated extraterrestrial conditions is therefore crucial for the success of future life detection missions. Ionizing radiation can be considered as the most deleterious factor for the long‐term preservation of potential biomarkers on Mars. Here, we report on the preservation potential of Raman signatures in the Antarctic strain CCCryo 231‐06 of the cyanobacterium Nostoc sp. after high doses of gamma irradiation. The carotenoids' signals, a well‐established biosignature model, dominate the Raman spectra at 532‐nm excitation wavelength due to resonance effects. But comparing their distribution and quantifying their preservation are still problematic in natural samples. To standardize the analyses, we successfully applied Raman mapping and signal‐to‐noise ratio masks to quantify the effects of irradiation. The typical in vivo Raman signatures of carotenoids could be detected after exposure to up to 56 kGy with significant deterioration in terms of signal coverage and signal‐to‐noise ratio. But they remained stable even after the highest dose of γ rays (117 kGy) tested in this study for colonies embedded in two different Martian mineral analogues. Data gathered during these ground‐based irradiation experiments contribute to interpret results from space experiments and will guide our search for life on Mars and other bodies of interest.
By investigating the survival and the biomarker detectability of a rock-inhabiting cyanobacterium, Chroococcidiopsis sp. CCMEE 029, the BIOMEX space experiment might contribute to a future exploitation of the Moon as a test-bed for key astrobiology tasks such as the testing of life-detection technologies and the study of life in space. Post-flight analyses demonstrated that the mixing of dried cells with sandstone and a lunar regolith simulant provided protection against space UV radiation. During the space exposure, dried cells not mixed with minerals were killed by 2.05 × 10 ² kJ m ⁻² of UV radiation, while cells mixed with sandstone or lunar regolith survived 1.59 × 10 ² and 1.79 × 10 ² kJ m ⁻² , respectively. No differences in survival occurred among cells mixed and not mixed with minerals and exposed to space conditions in the dark; this finding suggests that space vacuum and 0.5 Gy of ionizing radiation did not impair the cells’ presence in space. The genomic DNA of dead cells was severely damaged but still detectable with PCR amplification of a short target, thus suggesting that short sequences should be targeted in a PCR-based approach when searching for traces of life. The enhanced stability of genomic DNA of dried cells mixed with minerals and exposed to space indicates that DNA might still be detectable after prolonged periods, possibly up to millions of years in microbes shielded by minerals. Overall, the BIOMEX results contribute to future experiments regarding the exposure of cells and their biomarkers to deep space conditions in order to further test the lithopanspermia hypothesis, the biomarker stability and the microbial endurance, with implications for planetary protection and to determine if the Moon has been contaminated during past human missions.
PART I: RESISTANCE OF CHROOCOCCIDIOPSIS SPP. TO SPACE AND MARS-LIKE CONDITIONS - Chapter 1: Introducing Part I - Chapter 2: Survival and biosignature detection of Chroococcidiopsis spp. after ground-based simulations of extraterrestrial environments - Chapter 3: Enhanced resistance of Chroococcidiopsis biofilms, compared to their planktonic counterparts, to space and simulated Martian conditions in low Earth orbit - Chapter 4: Responses of the desert cyanobacterium Chroococcidiopsis to simulated Martian conditions in low Earth orbit: implications for the limits of terrestrial life and the habitability of Mars --------------------------------------------- PART II: CYANOBACTERIUM-BASED LIFE-SUPPORT SYSTEMS FOR THE FUTURE OF CREWED SPACE EXPLORATION - Chapter 5: Introducing Part II - Chapter 6: Cyanobacterium biomass as a substrate for heterotrophic growth on Mars
Chroococcidiopsis were exposed to low Earth conditions by using the EXPOSE-R2 facility outside the International Space Station. During the space mission, samples in Tray 1 (space vacuum and solar radiation, from λ ≈ 110 nm) and Tray 2 (Mars-like UV flux, λ > 200 nm and Mars-like atmosphere) received total UV (200–400 nm) fluences of about 4.58 × 102 kJ/m² and 4.92 × 102 kJ/m², respectively, and 0.5 Gy of cosmic ionizing radiation. Postflight analyses were performed on 2.5-year-old samples due to the space mission duration, from launch to sample return to the lab. The occurrence of survivors was determined by evaluating cell division upon rehydration and damage to the genome and photosynthetic apparatus by polymerase chain reaction–stop assays and confocal laser scanning microscopy. Biofilms recovered better than their planktonic counterparts, accumulating less damage not only when exposed to UV radiation under space and Mars-like conditions but also when exposed in dark conditions to low Earth conditions and laboratory control conditions. This suggests that, despite the shielding provided by top-cell layers being sufficient for a certain degree of survival of the multilayered planktonic samples, the enhanced survival of biofilms was due to the presence of abundant extracellular polymeric substances and to additional features acquired upon drying.
Outer membrane vesicles (OMVs), produced by nonpathogenic Gram-negative bacteria, have potentially useful biotechnological applications in extraterrestrial extreme environments. However, their biological effects under the impact of various stressors have to be elucidated for safety reasons. In the spaceflight experiment, model biofilm kombucha microbial community (KMC) samples, in which Komagataeibacter intermedius was a dominant community-member, were exposed under simulated Martian factors (i.e., pressure, atmosphere, and UV-illumination) outside the International Space Station (ISS) for 1.5 years. In this study, we have determined that OMVs from post-flight K. intermedius displayed changes in membrane composition, depending on the location of the samples and some other factors. Membrane lipids such as sterols, fatty acids (FAs), and phospholipids (PLs) were modulated under the Mars-like stressors, and saturated FAs, as well as both short-chain saturated and trans FAs, appeared in the membranes of OMVs shed by both post-UV-illuminated and "dark" bacteria. The relative content of zwitterionic and anionic PLs changed, producing a change in surface properties of outer membranes, thereby resulting in a loss of interaction capability with polynucleotides. The changed composition of membranes promoted a bigger OMV size, which correlated with changes of OMV fitness. Biochemical characterization of the membrane-associated enzymes revealed an increase in their activity (DNAse, dehydrogenase) compared to wild type. Other functional membrane-associated capabilities of OMVs (e.g., proton accumulation, interaction with linear DNA, or synaptosomes) were also altered after exposure to the spaceflight stressors. Despite alterations in membranes, vesicles did not acquire endotoxicity, cytotoxicity, and neurotoxicity. Podolich et al. Fitness of Post-stress Outer Membrane Vesicles Frontiers in Microbiology | www.frontiersin.org 2
In the context of astrobiological exposure and simulation experiments in the BIOMEX project, the lichen Circinaria gyrosa was investigated by Raman microspectroscopy. Owing to the symbiotic nature of lichens and their remarkable extremotolerance, C. gyrosa represents a valid model organism in recent and current astrobiological research. Biogenic compounds of C. gyrosa were studied that may serve as biomarkers in Raman assisted remote sensing missions, e.g. ExoMars. The surface as well as different internal layers of C. gyrosa have been characterized and data on the detectability and distribution of β-carotene, chitin and calcium oxalate monohydrate (whewellite) are presented in this study. Raman microspectroscopy was applied on natural samples and thin sections. Although calcium oxalates can also be formed by rare geological processes it may serve as a suitable biomarker for astrobiological investigations. In the model organism C. gyrosa, it forms extracellular crystalline deposits embedded in the intra-medullary space and its function is assumed to balance water uptake and gas exchange during the rare, moist to wet environmental periods that are physiologically favourable. This is a factor that was repeatedly demonstrated to be essential for extremotolerant lichens and other organisms. Depending on the decomposition processes of whewellite under extraterrestrial environmental conditions, it may not only serve as a biomarker of recent life, but also of past and fossilized organisms.
Introduction Raman spectra will be measured with the Raman Laser Spectrometer (RLS) onboard ExoMars in 2018 to identify organic compounds and mineral products as an indication of former or recent biological activi-ty. Investigation with the same specifications as those onboard the ExoMars mission is conducted to test the potential of identifying biological material on martian analogue material with Raman spectroscopy. Appropriate parameters concerning integration time and number of repititions for the detection of biological matter as well as for the determination of the mineral composition will be derived. In addition, problems are reported on using Raman spectroscopy to discriminate the microorganisms from the mineral background. Biological sample Cyanobacteria and methane producing archaea are chosen to represent potential life on Mars. Prokaryotes like archaea and bacteria appeared on early Earth at least 3.8 to 3.5 billion years ago (Gya). Life might have developed under similar conditions on Mars as on Earth or might have been transferred from Earth (or vice versa). At that time on Mars the climate was more temperate and wet compared to the present day as inferred from geological evidence for liquid water on the ancient martian surface. Methane is known to be present on Mars. A source is still unknown. Methane might originate from geothermal or biological activities nearby the surface of the red planet. Cyanobacteria and prokaryotes using photosystem I use pigments such as scytonemin and beta-carotene as UV protection. Especially beta - carotene emits a strong Raman signal at the applied laser excitation wavelength. Raman measurements are used for detection of coccid, chain, and biofilm forming cyanobacteria Nostoc commune strain 231-06 (Fraunhofer IMBT CCCryo) on the below described Mars analogue mineral mixtures. Nostoc commune is known to be resistant to desiccation, UV B radiation and low temperatures, and thus suitable as a candidate for a potential life form on Mars. Furthermore, the Raman technique is applied on samples of the methane producing archaea candidatus Methanosarcina gelisolum (strain SMA 21) isolated from Siberian permafrost affected soils and on these archaea embedded in the martian analogue material. Martian analogue material In this investigation two different Mars analogue materials prepared from mineral and rock mixtures are used. The (1) Phyllosilicatic Mars Regolith Simulant (P-MRS) and (2) Sulfatic Mars Reg-olith Simulant (S-MRS) reflect the current understanding regarding environmental changes on Mars. Weathering or hydrothermal alteration of crustal rocks and of secondary mineralization during part of the Noachian and Hesperian epoch followed by the prevailing cold and dry oxidising condition with formation of anhydrous iron oxides. The use of two different mixtures accounts for the observations that phyllosilicatic deposits do not occur together with sulphatic deposits. P-MRS and S-MRS serve as the analogue geomaterials in which the cells of cyanobacteria and of methanogenes are embedded. Results Varying periods of measurement time and number of repetitions are used to get optimal Raman spectra for cyanobacteria and methanogenes. If cyanobacteria are present, beta-carotene is the dominant feature in the spectrum. Measurement times need to be adjusted to obtain optimal spectra of the P-MRS and S-MRS with cyanobacteria. Measurements performed with various values of measurement time and number of measurements show clearly the improvement achieved by increasing the time per spectrum from 1s to 20s. But it is desirable to find a set of small values of measurement time and number of repetitions in order to optimize the detection of minerals and biological markers and to reduce the disturbing effect of cosmic rays. A measurement regime is proposed for mineral mixtures with cyanobacteria on the basis of the RLS instrument characteristics: A procedure on ExoMars should start with a measurement time of only a few seconds to identify both biomarkers and minerals. If no biomarkers can be identified the time and number of measurements need to be increased until spectra of minerals are obtained. The measurement time should be selected between 1s (for b-carotene) and 20s (for minerals) for a laser power of 1mW (spot diameter < 2 µm). Future investigations of Raman measurement parameters should consider the different environmental parameters on Mars like atmospheric pressure, composition and temperature. For methanogens a different measurement regime needs to be developed. Raman analytics are capable to identify biosignatures like beta – carotene on a multi-mineral mixture similar to those expected to be encountered during the ExoMars mission.
The BIOMEX (BIOlogy and Mars Experiment) is part of the European Space Agency (ESA) space mission EXPOSE-R2 in Low-Earth Orbit, devoted to exposing microorganisms for 1.5 years to space and simulated Mars conditions on the International Space Station. In preparing this mission, dried colonies of the Antarctic cryptoendolithic black fungus Cryomyces antarcticus CCFEE 515, grown on martian and lunar analog regolith pellets, were subjected to several ground-based preflight tests, Experiment Verification Tests, and Science Verification Tests (SVTs) that were performed to verify (i) the resistance of our model organism to space stressors when grown on extraterrestrial rock analogues and (ii) the possibility of detecting biomolecules as potential biosignatures. Here, the results of the SVTs, the last set of experiments, which were performed in ultraviolet radiation combined with simulated space vacuum or simulated martian conditions, are reported. The results demonstrate that C. antarcticus was able to tolerate the conditions of the SVT experiment, regardless of the substratum in which it was grown. DNA maintained high integrity after treatments and was confirmed as a possible biosignature; melanin, which was chosen to be a target for biosignature detection, was unambiguously detected by Raman spectroscopy.
Lichens are extremely resistant organisms that colonize harsh climatic areas, some of them defined as “Mars-analog sites.” There still remain many unsolved questions as to how lichens survive under such extreme conditions. Several studies have been performed to test the resistance of various lichen species under space and in simulated Mars-like conditions. The results led to the proposal that Circinaria gyrosa (Lecanoromycetes, Ascomycota) is one of the most durable astrobiological model lichens. However, although C. gyrosa has been exposed to Mars-like environmental conditions while in a latent state, it has not been exposed in its physiologically active mode. We hypothesize that the astrobiological test system “Circinaria gyrosa,” could be able to be physiologically active and to survive under Mars-like conditions in a simulation chamber, based on previous studies performed at dessicated-dormant stage under simulated Mars-like conditions, that showed a complete recover of the PSII activity (Sánchez et al., 2012). Epifluorescence and confocal laser scanning microscopy (CLSM) showed that living algal cells were more abundant in samples exposed to niche conditions, which simulated the conditions in micro-fissures and micro-caves close to the surface that have limited scattered or time-dependent light exposure, than in samples exposed to full UV radiation. The medulla was not structurally affected, suggesting that the niche exposure conditions did not disturb the lichen thalli structure and morphology as revealed by field emission scanning electron microscopy (FESEM). In addition, changes in the lichen thalli chemical composition were determined by analytical pyrolysis. The chromatograms resulting from analytical pyrolysis at 500°C revealed that lichen samples exposed to niche conditions and full UV radiation consisted primarily of glycosidic compounds, lipids, and sterols, which are typical constituents of the cell walls. However, specific differences could be detected and used as markers of the UV-induced damage to the lichen membranes. Based on its viability responses after rehydration, our study shows that the test lichen survived the 30-day incubation in the Mars chamber particularly under niche conditions. However, the photobiont was not able to photosynthesize under the Mars-like conditions, which indicates that the surface of Mars is not a habitable place for C. gyrosa.
Kombucha is a multispecies microbial community which produce bacterial cellulose — a polymer molecule to be a candidate for a biomarker of life. For the pre-flight ground-based phase of Biology and Mars Experiment (BIOMEX), the multi-microbial cellulose-based biofilm was embedded in mineral material to test the structural integrity of the bacterial cellulose and a survival of community-members under Mars-like CO 2-rich atmosphere, pressure and solar irradiation spectrum similar to that on the surface of Mars. During the preparatory testing stage it was found that after the synergistic action of a set of stressful space-and Mars-associated factors the mineralized cellulose preserved the characteristic molecular fingerprints, which might be detected instrumentally. The flight stage of the BIOMEX begun on July, 2014 and will last for 12—18 months on the EXPOSE-R2 platform mounted by the astronauts outside the ISS.
Introducing of the DNA metabarcoding analysis of probiotic microbial communities allowed getting insight into their functioning and establishing a better control on safety and efficacy of the probiotic communities. In this work the kombucha poly-microbial probiotic community was analysed to study its flexibility under different growth conditions. Environmental DNA sequencing revealed a complex and flexible composition of the kombucha microbial culture (KMC) constituting more bacterial and fungal organisms in addition to those found by cultural method. The community comprised bacterial and yeast components including cultured and uncultivable microorganisms. Culturing the KMC under different conditions revealed the core part of the community which included acetobacteria of two genera Komagataeibacter (former Gluconacetobacter) and Gluconobacter, and representatives of several yeast genera among which Brettanomyces/Dekkera and Pichia (including former Issatchenkia) were dominant. Herbaspirillum spp. and Halomonas spp., which previously had not been described in KMC, were found to be minor but permanent members of the community. The community composition was dependent on the growth conditions. The bacterial component of KMC was relatively stable, but may include additional member—lactobacilli. The yeast species composition was significantly variable. High-throughput sequencing showed complexity and variability of KMC that may affect the quality of the probiotic drink. It was hypothesized that the kombucha core community might recruit some environmental bacteria, particularly lactobacilli, which potentially may contribute to the fermentative capacity of the probiotic drink. As many KMC-associated microorganisms cannot be cultured out of the community, a robust control for community composition should be provided by using DNA metabarcoding.
Forty years after the Viking missions, International space agencies are ready to resume the search for life on Mars (and in our Solar System). Indeed, new instruments are able to detect traces of extant or extinct life. They will be sent to Mars onboard the two next rovers: ExoMars2020 and Mars2020. Among them, instruments based on Raman spectroscopy are very promising thanks to their capacity to identify both the mineralogical context and organic molecules of potential biogenic origin. However, in order to support these future missions, it is very important to investigate the degree of preservation and the evolution of potential biosignatures under simulated and real space conditions by Raman spectroscopy. To this end, the BIOMEX (BIOlogy and Mars EXperiment) experiment aims at investigating the endurance of extremophiles and stability of biomolecules under space and Mars-like conditions in the presence of Martian mineral analogues (de Vera et al. 2012). BIOMEX was part of the EXPOSE-R2 mission of the European Space Agency which allowed a 15-month exposure, on the outer side of the International Space Station, which comprises also three other astrobiology experiments between July 2014 and February 2016. Among the potential biosignatures investigated, the photoprotective carotenoid pigments (present either in photosynthetic organisms such as plants, algae, cyanobacteria and in some bacteria and archaea) have been classified as high priority targets for biomolecule detection on Mars and therefore used as a model biosignature due to their stability and easy identification by Raman spectroscopy (Böttger et al. 2012). We report here on the first results from the analysis of two carotenoids containing organisms: the cyanobacterium Nostoc sp. (strain CCCryo 231-06; = UTEX EE21 and CCMEE 391) isolated from Antarctica and the green alga cf. Sphaerocystis sp. (strain CCCryo 101-99) isolated from Spitsbergen. Desiccated cells of these organisms were exposed to space conditions and to simulated Mars-like conditions in space. They were cultured on Martian mineral analogues (phyllosilicatic and sulfatic Mars regolith simulants) and a Lunar regolith analogue and analyzed with a 532nm Raman spectroscope operating at 1mW laser power. Carotenoids in both organisms were surprisingly still detectable at relatively high levels after being exposed for 15 months in Low Earth Orbit to UV, cosmic rays, vacuum (or Mars-like atmosphere) and temperatures stresses regardless of the mineral matrix used. Further analyses will help us to correlate these results with survival potential, cellular damages or stability and the different extremophiles tested in the BIOMEX experiment.
The BIOMEX (BIOlogy and Mars EXperiment) experiment aims at investigating the endurance of extremophiles and stability of biomolecules under space and Mars-like conditions in the presence of Martian mineral analogues (de Vera et al. 2012). To this end, extensive ground-based simulation studies and a space experiment were performed. Indeed, BIOMEX was part of the EXPOSE-R2 mission of the European Space Agency which allowed a 15-month exposure, on the outside of the International Space Station, of four astrobiology experiments between July 2014 and February 2016. The preservation and evolution of Raman biosignatures under real space conditions is of particular interest for guiding future search-for-life missions to Mars (and other planetary objects) carrying Raman spectrometers (such as the Raman Laser Spectrometer instrument on board the future ExoMars rover). Among the potential biosignatures investigated, the photoprotective carotenoid pigments (present either in photosynthetic organisms such as plants, algae, cyanobacteria and in some bacteria and archaea) have been classified as high priority targets for biomolecule detection on Mars and therefore used as biosignature models due to their stability and easy identification by Raman spectroscopy (Böttger et al. 2012). We report here on the first results from the analysis of two carotenoids containing organisms: the cyanobacterium Nostoc sp. (strain CCCryo 231-06; = UTEX EE21 and CCMEE 391) isolated from Antarctica and the green alga cf. Sphaerocystis sp. (strain CCCryo 101-99) isolated from Spitsbergen. Desiccated cells of these organisms were exposed to space and simulated Mars-like conditions in space in the presence of two Martian mineral analogues (phyllosilicatic and sulfatic Mars regolith simulants) and a Lunar regolith analogue and analyzed with a 532nm Raman microscope at 1mW laser power. Carotenoids in both organisms were surprisingly still detectable at relatively high levels after being exposed for 15 months in Low Earth Orbit to UV, cosmic rays, vacuum (or Mars-like atmosphere) and temperatures stresses regardless of the mineral matrix used. Further analyses will help us to correlate these results with survival potential, cellular damages or stability and the different extremophiles tested in the BIOMEX experiment.
The Antarctic black meristematic fungus Cryomyces antarcticus CCFEE 515 occurs endolithically in the McMurdo Dry Valleys of Antarctica, one of the best analogue for Mars environment on Earth. To date, this fungus is considered one of the best eukaryotic models for astrobiological studies and has been repeatedly selected for space experiments in the last decade. The obtained results are reviewed here, with special focus on responses to space relevant irradiation, UV radiation, and both sparsely and densely ionizing radiation, which represent the major injury for a putative space-traveller. The remarkable resistance of this model organism to space stress, its radioresistance in particular, and mechanisms involved, significantly contributed to expanding our concept of limits for life and provided new insights on the origin and evolution of life in planetary systems, habitability, and biosignatures for life detection as well as on human protection during space missions.
Biofilm-forming microbial communities are known as the most robust assemblages that can survive in harsh environments. Biofilm-associated microorganisms display greatly increased resistance to physical and chemical adverse conditions, and they are expected to be the first form of life on Earth or anywhere else. Biological molecules synthesized by biofilm -protected microbiomes may serve as markers of the nucleoprotein life. We offer a new experimental model, a kombucha multimicrobial culture (KMC), to assess a structural integrity of a widespread microbial polymer – cellulose – as a biosignature of bacteria-producers for the multipurpose international project “BIOlogical and Mars Experiment (BIOMEX)”, which aims to study the vitality of pro- and eukaryotic organisms and the stability of organic biomolecules in contact with minerals to analyze the detectability of life markers in the context of a planetary background. In this study, we aimed to substantiate the detectability of mineralized cellulose with spectroscopy and to study the KMC macrocolony phenotype stability under adverse conditions (UV, excess of inorganics etc.). Cellulose matrix of the KMC macrocolony has been mineralized in the mineral-water interface under assistance of KMC-members. Effect of bioleached ions on the cellulose matrix has been visible, and the FT-IR spectrum proved changes in cellulose structure. However, the specific cellulose band vibration, confirming the presence of β(1,4)-linkages between monomers, has not been quenched by secondary minerals formed on the surface of pellicle. The cellulose-based KMC macrocolony phenotype was in a dependence on extracellular matrix components (ionome, viriome, extracellular membrane vesicles), which provided its integrity and rigidness in a certain extent under impact of stressful factors.
The Low Earth Orbit (LEO) experiment Biology and Mars Experiment (BIOMEX) is an interdisciplinary and international space research project selected by ESA. The experiment will be accommodated on the space exposure facility EXPOSE-R2 on the International Space Station (ISS) and is foreseen to be launched in 2013. The prime objective of BIOMEX is to measure to what extent biomolecules, such as pigments and cellular components, 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 building a biosignature data base (e.g. the proposed creation of an international Raman library). The library will be highly relevant for future space missions such as the search for life on Mars. The secondary scientific objective is to analyze to what extent terrestrial extremophiles are able to survive in space and to determine which interactions between biological samples and selected minerals (including terrestrial, Moon- and Mars analogs) can be observed under space and Mars-like conditions. In this context, the Moon will be an additional platform for performing similar experiments with negligible magnetic shielding and higher solar and galactic irradiation compared to LEO. Using the Moon as an additional astrobiological exposure platform to complement ongoing astrobiological LEO investigations could thus enhance the chances of detecting organic traces of life on Mars. We present a lunar lander mission with two related objectives: a lunar lander equipped with Raman and PanCam instruments which can analyze the lunar surface and survey an astrobiological exposure platform. This dual use of testing mission technology together with geo- and astrobiological analyses will significantly increase the science return, and support the human preparation objectives. It will provide knowledge about the Moon′s surface itself and, in addition, monitor the stability of life-markers, such as cells, cell components and pigments, in an extraterrestrial environment with much closer radiation properties to the surface of Mars. The combination of a Raman data base of these data together with data from LEO and space simulation experiments, will lead to further progress on the analysis and interpretation of data that we will obtain from future Moon and Mars exploration missions.
The most resistant cyanobacteria can be found in tropic deserts and in polar and alpine habitats. The reason for their resistance can be explained by their occurrence in intensely irradiated, very dry and/or cold environments which are supposed to be as close as possible similar to Martian surface conditions. A systematically approach comparing measurements on photosynthetic activity of cyanobacteria in relation to measured environmental parameters obtained in Mars analog field sites with data collected from space exposed samples or during Mars simulation experiments will show differences and common results after analyzing the investigated organisms. Some of the investigated species are foreseen to be exposed during the next ESA-space-exposure experiment BIOMEX either directly to real space conditions on space exposure platforms like EXPOSE-R2 on the International Space Station or to Mars simulation conditions in a Mars simulation chamber. Some of the species were still exposed to both of the extreme environmental conditions and some of the results will be presented and might serve for future investigations as references. We will emphasize that in parallel monitoring of environmental parameters on Mars analog field sites was performed as well as partly in space and in the simulation chambers. This experimental combination might help to get a better impression about the influence of each of the tested parameters on metabolic activity of the tested cyanobacteria in complete different planetary environments comparing characterized habitats on our home planet Earth with those we might expect according to recently observed data on Mars. The outcome of this work could be relevant to classify e.g. Mars as a habitable planet by a new combination of different experimental and biological approaches and to evaluate and discuss the likelihood of terra forming Mars in the far future.
This article provides first insights into some of the fascinating aspects of astrobiology. The central focus of this research theme is directed towards questions which have interested humans for millennia: How has life developed? Where do we come from? Are we alone in the Universe? In order to approach these questions, astrobiology brings together a variety of disciplines such as astronomy, astrophysics, biology, biochemistry, chemistry, geology, mineralogy, and cosmology. In particular, scientists are more and more interested into the biological aspects and the interactions of (micro)-organisms with their geological environment. This interest is further fueled by numerous NASA and ESA missions to Mars that have spawned new insights into Mars as a potential habitat for life. Earth analog environments, which are characterized by conditions that occur in other parts of our solar system in even more extreme forms play an important role for astrobiological research. Examples include habitats that are characterized by extreme dryness and/or coldness such as the Atacama Desert in Chile, liquid asphalt lakes in Trinidad or the permafrost areas in Siberia. Extremophilic microorganisms such as cyanobacteria and methanogenic archaea or higher organisms such as lichens and bryophytes are used as model organisms to study the limits of life under simulated extreme conditions. In recent years, an active research network has been established in the Berlin-Brandenburg region that carried out joint astrobiology and habitability studies and which among other projects is currently conducting an ESA experiment onboard the International Space Station ISS.
Lichens are extremophile organisms, they live in the most extreme conditions, colonizing areas with extreme temperatures, high aridity condition and high UV-radiation. Therefore they have been by far the most successful settlers of the Antarctic continent. Also in the laboratory they survive temperatures near the absolute cero and absolute dryness without difficulty. Lichen species have distinct likes and dislikes when it come to the physico-chemical properties of the substrate while the group of lichens as a whole is pretty adaptable to vari- ous substrata (from rocks to glass). The main feature/aspect of their evolutionary/ecological success of this capacity is the close symbiotic relation between two organisms, a fungi and a cyanobacteria or an algae , allowing them to survive at real space  and at Mars con- ditions [3, 4, 5], such as that on the ISS. At the exposure platform EXPOSE-R2 on ISS (2014-2016), samples of the lichen species Circinaria gyrosa belonging to the BIOMEX ex- periment (Biology and Mars Experiment, ESA) , were exposed during 18 months to real space and to a Mars simulated environment to study Mars habitability and resistance to real space conditions. Also the identification of biomarkers was done to include them as reference for future space missions to Mars (Exo Mars). After the return of the mission at June 2016, the first preliminary analysis were performed, showing the metabolic activity a quick and complete recovery of the dark space control samples exposed to space vacuum and Mars-like atmosphere. In contrast, the samples directly exposed to space radiation showed slow recovery in reference to their observed original activity. Electron and fluores- cence microscopy techniques also revealed that the viability of C. gyrosa exposed to space conditions decreased in comparison to those exposed to Mars-like environment. Moreover, differences were observed between samples positioned at level 1 and level 2. In general, TEM and FESEM observations showed that samples at level 2 (basal samples) were slightly affected in their morphology/ultrastructure by the exposure conditions. In contrast, cellular ultrastructure alterations were clearly evident for samples exposed to space radiation, which led to a shrinkage process. The cell walls were irregularly shaped and debris of the major organelles were visible. Now, the biomolecular changes of the DNA are in study by PCR and sequencing techniques. In contrast to these studies, the biogeochemical variations will be examined with spectroscopic analyses (Raman) to look for possible degradation of cell surfaces and pigments which were in contact with terrestrial rocks, and Martian analogue regolith. These experiments will contribute to answer questions on the habitability of Mars, on the likelihood of the Lithopanspermia HYPOTHESIS y  and will be of relevance for planetary protection issues.
Future search-for-life missions to Mars will carry for the first time Raman spectrometers (such as the Raman Laser Spectrometer instrument aboard the future ExoMars rover) for in situ analysis of extraterrestrial material (see e.g. Rull et al., 2010). Raman spectroscopy is indeed a powerful and non-destructive technique that can provide structural molecular information from both organic (potential biosignatures) and inorganic (mineral environment) samples. The preservation and evolution of Raman biosignatures under simulated extraterrestrial conditions is therefore of particular interest for guiding these future missions and interpret future data. Among the damaging factors present in extraterrestrial environments and in particular on Mars, ionizing radiations can be considered the most deleterious for the long term preservation of such markers (Dartnell et al., 2012). Cyanobacterial photoprotective pigments (namely carotenoids) have been classified as high priority targets for biomolecule detection on Mars and therefore used as biosignature models due to their stability and easy identification by Raman spectroscopy (Böttger et al., 2012). We report here on their preservation potential in the cyanobacterium Nostoc commune strain 231–06 (Fraunhofer IMBT CCCryo) after high dose γ-ray irradiation performed in the frame of the STARLIFE project (Moeller et al., 2016). The typical Raman signatures of carotenoids could be detected after up to 56 kGy of γ-rays on colonies irradiated alone but with significant deterioration in terms of signal coverage and peak heights. However, for colonies embedded in two different Martian mineral analogues (phyllosilicatic and sulfatic Mars regolith simulants), carotenoids signatures remained detectable even at the highest tested dose of γ-rays (117kGy) with no significant effect on the signal coverage and only slight decrease of peak heights. Data gathered during these ground-based irradiation experiments will contribute to interpret future results from space experiments and guide our search for life on Mars and other bodies of interest. References: Böttger, U., de Vera, J.-P., Fritz, J., Weber, I., Hübers, H.-W. & Schulze-Makuch, D. (2012) Optimizing the detection of carotene in cyanobacteria in a martian regolith analogue with a Raman spectrometer for the ExoMars mission. Planetary and Space Science 60:356–362. Dartnell, L.R., Page, K., Jorge-Villar, S.E., Wright, G., Munshi, T., Scowen, I.J., Ward, J.M. & Edwards, H.G.M. (2012) Destruction of Raman biosignatures by ionising radiation and the implications for life detection on Mars. Analytical and Bioanalytical Chemistry 403:131–144. Moeller, R., Raguse, M., Leuko, S., Berger, T., Elisabeth Hellweg, C., Fujimori, A., Okayasu, R., Horneck, G. & the STARLIFE research group (2016) STARLIFE – an international campaign to study the role of galactic cosmic radiation in astrobiological model system. Astrobiology (under revision; AST-2015-1452). Rull, F., Sansano, A., Díaz, E., Canora, C.P., Moral, A.G., Tato, C., Colombo, M., Belenguer, T., Fernández, M., Manfredi, J.A.R., Canchal, R., Dávila, B., Jiménez, A., Gallego, P., Ibarmia, S., Prieto, J.A.R., Santiago, A., Pla, J., Ramos, G. & González, C. (2010) ExoMars Raman laser spectrometer overview. (Vol. 7819). Presented at the Instruments, Methods, and Missions for Astrobiology XIII, San Diego, California: SPIE proceedings.
Outer space, the final frontier, is a hostile and unforgiving place for any form of life as we know it. The unique environment of space allows for a close simulation of Mars surface conditions that cannot be simulated as accurately on the Earth. For this experiment, we tested the resistance of Deinococcus radiodurans to survive exposure to simulated Mars-like conditions in low-Earth orbit for a prolonged period of time as part of the Biology and Mars experiment (BIOMEX) project. Special focus was placed on the integrity of the carotenoid deinoxanthin, which may serve as a potential biomarker to search for remnants of life on other planets. Survival was investigated by evaluating colony forming units, damage inflicted to the 16S rRNA gene by quantitative PCR, and the integrity and detectability of deinoxanthin by Raman spectroscopy. Exposure to space conditions had a strong detrimental effect on the survival of the strains and the 16S rRNA integrity, yet results show that deinoxanthin survives exposure to conditions as they prevail on Mars. Solar radiation is not only strongly detrimental to the survival and 16S rRNA integrity but also to the Raman signal of deinoxanthin. Samples not exposed to solar radiation showed only minuscule signs of deterioration. To test whether deinoxanthin is able to withstand the tested parameters without the protection of the cell, it was extracted from cell homogenate and exposed to high/low temperatures, vacuum, germicidal UV-C radiation, and simulated solar radiation. Results obtained by Raman investigations showed a strong resistance of deinoxanthin against outer space and Mars conditions, with the only exception of the exposure to simulated solar radiation. Therefore, deinoxanthin proved to be a suitable easily detectable biomarker for the search of Earth-like organic pigment-containing life on other planets.
The BIOlogy and Mars Experiment (BIOMEX) is part of the European Space Agency (ESA) space mission EXPOSE-R2 in Low Earth Orbit (LEO), aiming to expose microorganisms for 1.5 years to space and simulated Mars-like conditions on the International Space Station (ISS). In preparation of this mission, dried colonies of the Antarctic cryptoendolithic black fungus Cryomyces antarcticus CCFEE 515, grown on Martian and Lunar analogue regolith pellets, were subjected to several ground-based preflight tests, Experiment Verification Tests (EVTs) and Science Verification Tests (SVTs). These tests aimed to verify i) the resistance of our model organism to space stressors when grown on extraterrestrial rock analogues and ii) the possibility to detect biomolecules as potential biosignatures in reference to support the Exomars 2020 mission. Here some results are reported showing the outcome of the SVTs, the last set of experiments, where the effect of UV radiation was analyzed if combined with simulated space vacuum or simulated Mars-like conditions. The analyses performed by Gas Chromatography-Mass Spectrometry showed the presence of fungal metabolites, as azelaic acid, that remain unaltered after the different expositions and treatments. In addition first results of Raman Spectroscopy analysis on melanin will be presented. Further investigation is necessary to derive the appropriate parameter set for Raman spectroscopy of melanin. Transmission Electron Microscopy (TEM) observations showed different results in preservation of cell’s ultrastructure.
The space environment is regularly used for experiments addressing astrobiology research goals. The specific conditions prevailing in Earth orbit and beyond, notably the radiative environment (photons and energetic particles) and the possibility to conduct long-duration measurements, have been the main motivations for developing experimental concepts to expose chemical or biological samples to outer space, or to use the reentry of a spacecraft on Earth to simulate the fall of a meteorite. This paper represents an overview of past and current research in astrobiology conducted in Earth orbit and beyond, with a special focus on ESA missions such as Biopan, STONE (on Russian FOTON capsules) and EXPOSE facilities (outside the International Space Station). The future of exposure platforms is discussed, notably how they can be improved for better science return, and how to incorporate the use of small satellites such as those built in cubesat format.
Kombucha microbial community (KMC) produces a cellulose-based biopolymer of industrial importance and a probiotic beverage. KMC-derived cellulose-based pellicle film is known as a highly adaptive microbial macrocolony - a stratified community of prokaryotes and eukaryotes. In the framework of the multipurpose international astrobiological project "BIOlogy and Mars Experiment (BIOMEX)," which aims to study the vitality of prokaryotic and eukaryotic organisms and the stability of selected biomarkers in low Earth orbit and in a Mars-like environment, a cellulose polymer structural integrity will be assessed as a biomarker and biotechnological nanomaterial. In a preflight assessment program for BIOMEX, the mineralized bacterial cellulose did not exhibit significant changes in the structure under all types of tests. KMC members that inhabit the cellulose-based pellicle exhibited a high survival rate; however, the survival capacity depended on a variety of stressors such as the vacuum of space, a Mars-like atmosphere, UVC radiation, and temperature fluctuations. The critical limiting factor for microbial survival was high-dose UV irradiation. In the tests that simulated a 1-year mission of exposure outside the International Space Station, the core populations of bacteria and yeasts survived and provided protection against UV; however, the microbial density of the populations overall was reduced, which was revealed by implementation of culture-dependent and culture-independent methods. Reduction of microbial richness was also associated with a lower accumulation of chemical elements in the cellulose-based pellicle film, produced by microbiota that survived in the post-test experiments, as compared to untreated cultures that populated the film.
Scientists use the Earth as a tool for astrobiology by analyzing planetary field analogues (i.e. terrestrial samples and field sites that resemble planetary bodies in our Solar System). In addition, they expose the selected planetary field analogues in simulation chambers to conditions that mimic the ones of planets, moons and Low Earth Orbit (LEO) space conditions, as well as the chemistry occurring in interstellar and cometary ices. This paperreviews the ways the Earth is used by astrobiologists: (i) by conducting planetary field analogue studies to investigate extant life from extreme environments, its metabolisms, adaptation strategies and modern biosignatures; (ii) by conducting planetary field analogue studies to investigate extinct life from the oldest rocks on our planet and its biosignatures; (iii) by exposing terrestrial samples to simulated space or planetary environments and producing a sample analogue to investigate changes in minerals, biosignatures and microorganisms. The European Space Agency (ESA) created a topical team in 2011 to investigate recent activities using the Earth as a tool for astrobiology and to formulate recommendations and scientific needs to improve ground-based astrobiological research. Space is an important tool for astrobiology (see Horneck et al. in Astrobiology, 16:201–243, 2016; Cottin et al., 2017), but access to space is limited. Complementing research on Earth provides fast access, more replications and higher sample throughput. The major conclusions of the topical team and suggestions for the future include more scientifically qualified calls for field campaigns with planetary analogy, and a centralized point of contact at ESA or the EU for the organization of a survey of such expeditions. An improvement of the coordinated logistics, infrastructures and funding system supporting the combination of field work with planetary simulation investigations, as well as an optimization of the scientific return and data processing, data storage and data distribution is also needed. Finally, a coordinated EU or ESA education and outreach program would improve the participation of the public in the astrobiological activities.
Este trabajo es un review interdisciplinar que recoge la historia de 60 años de investigación astrobiológica en el espacio (desde el comienzo de la “Exobiología”, Lederberg 1960), con el fín de obtener respuestas sobre la posibilidad de encontrar vida extraterrestre, los efectos del ambiente espacial sobre microorganismos (ref. protección planetaria), el origen de la vida en la Tierra, y finalmente su soporte en futuras misiones de exploración. Para investigar estas questiones, en 2011 la ESA funda un Topical Team en Astrobiología, al que pertenezco. El objetivo de éste trabajo es aparte de mencionar los desarrollos y resultados más recientes, planificar experimentos novedosos en consonancia con las expectativas de la comunidad científica. Para ello se enfoca en la descripción de los beneficios de utilizar el ambiente espacial de radiación y la investigación realizada en el campo de la Astrobiología, usando como herramienta el espacio. Se exponen diferentes plataformas experimentales espaciales, hardware, y muestras usadas en los experimentos realizados. La publicación presenta primeramente las plataformas de exposición para experimentos químicos y biológicos, y después dos capítulos dedicados a un número seleccionado de experimentos específicos relacionados con la Química y con la Biología, respectivamente, usando algunas veces las facilities comunes expuestas anteriormente. El estudio propone la modificación de las plataformas pasivas espaciales para incluir experimentos más complejos con medidas en tiempo real, lo que es relevante para garantizar valor añadido similar al obtenido en experimentos de simulación en cámaras planetarias. Mi participación ha sido como co-autora del paper, aportando la descripción y resultados de los experimentos realizados en la plataforma BIOPAN de las misiones del satélite FOTON M2 y M3, en los que he participado como Co-I (Experimento LICHENS, 2005) y como IP (Experimento LITHOPANSPERMIA, 2007). Allí detallo los hardware y resultados para cada experimento. Finalmente se propone una perspectiva para futuros desarrollos en este campo de investigación y recomendaciones del Topical Team de Astrobiología a la ESA.
Methanogenic archaea from Siberian permafrost are suitable model organisms that meet many of the preconditions for survival on the martian subsurface. These microorganisms have proven to be highly resistant when exposed to diverse stress factors such as desiccation, radiation and other thermo-physical martian conditions. In addition, the metabolic requirements of methanogenic archaea are in principle compatible with the environmental conditions of the Red Planet. The ExoMars mission will deploy a rover carrying a Raman spectrometer among the analytical instruments in order to search for signatures of life and to investigate the martian geochemistry. Raman spectroscopy is known as a powerful nondestructive optical technique for biosignature detection that requires only little sample preparation. In this study, we describe the use of confocal Raman microspectroscopy (CRM) as a rapid and sensitive technique for characterization of the methanogenic archaeon Methanosarcina soligelidi SMA21 at the single cell level. These studies involved acquisition of Raman spectra from individual cells isolated from microbial cultures at different stages of growth. Spectral analyses indicated a high degree of heterogeneity between cells of individual cultures and also demonstrated the existence of growth‐phase specific Raman patterns. For example, besides common Raman patterns of microbial cells, CRM additionally revealed the presence of lipid vesicles and CaCO3 particles in microbial preparations of M. soligelidi SMA‐21, a finding that could be confirmed by electron microscopy. The results of this study suggest that heterogeneity and diversity of microorganisms have to be considered when using Raman‐based technologies in future space exploration missions.
Methanogenic archaea are widespread anaerobic microorganisms responsible for the production of biogenic methane. Several new species of psychrotolerant methanogenic archaea were recently isolated from a permafrost-affected soil in the Lena Delta (Siberia, Russia), showing an exceptional resistance against desiccation, osmotic stress, low temperatures, starvation, UV and ionizing radiation when compared to methanogens from non-permafrost environments. To gain a deeper insight into the differences observed in their resistance, we described the chemical composition of methanogenic strains from permafrost and non-permafrost environments using confocal Raman microspectroscopy (CRM). CRM is a powerful tool for microbial identification and provides fingerprint-like information about the chemical composition of the cells. Our results show that the chemical composition of methanogens from permafrost-affected soils presents a high homology and is remarkably different from strains inhabiting non-permafrost environments. In addition, we performed a phylogenetic reconstruction of the studied strains based on the functional gene mcrA to prove the different evolutionary relationship of the permafrost strains. We conclude that the permafrost methanogenic strains show a convergent chemical composition regardless of their genotype. This fact is likely to be the consequence of a complex adaptive process to the Siberian permafrost environment and might be the reason underlying their resistant nature.
Lichens are symbioses of two organisms, a fungal mycobiont and a photoautotrophic photobiont. In nature, many lichens tolerate extreme environmental conditions and thus became valuable models in astrobiological research to fathom biological resistance towards non-terrestrial conditions; including space exposure, hypervelocity impact simulations as well as space and Martian parameter simulations. All studies demonstrated the high resistance towards non-terrestrial abiotic factors of selected extremotolerant lichens. Besides other adaptations, this study focuses on the morphological and anatomical traits by comparing five lichen species-Circinaria gyrosa, Rhizocarpon geographicum, Xanthoria elegans, Buellia frigida, Pleopsidium chlorophanum-used in present-day astrobiological research. Detailed investigation of thallus organization by microscopy methods allows to study the effect of morphology on lichen resistance and forms a basis for interpreting data of recent and future experiments. All investigated lichens reveal a common heteromerous thallus structure but diverging sets of morphological-anatomical traits, as intra-/extra-thalline mucilage matrices, cortices, algal arrangements, and hyphal strands. In B. frigida, R. geographicum, and X. elegans the combination of pigmented cortex, algal arrangement, and mucilage seems to enhance resistance, while subcortex and algal clustering seem to be crucial in C. gyrosa, as well as pigmented cortices and basal thallus protrusions in P. chlorophanum. Thus, generalizations on morphologically conferred resistance have to be avoided. Such differences might reflect the diverging evolutionary histories and are advantageous by adapting lichens to prevalent abiotic stressors. The peculiar lichen morphology demonstrates its remarkable stake in resisting extreme terrestrial conditions and may explain the high resistance of lichens found in astrobiological research.
Lichens, which are symbioses of a fungus and one or two photoautotrophs, frequently tolerate extreme environmental conditions. This makes them valuable model systems in astrobiological research to fathom the limits and limitations of eukaryotic symbioses. Various studies demonstrated the high resistance of selected extremotolerant lichens towards extreme, non-terrestrial abiotic factors including space exposure, hypervelocity impact simulations as well as space and Martian parameter simulations. This study focusses on the diverse set of secondary lichen compounds (SLCs) that act as photo- and UVR-protective substances. Five lichen species used in present-day astrobiological research were compared: Buellia frigida, Circinaria gyrosa, Rhizocarpon geographicum, Xanthoria elegans, and Pleopsidium chlorophanum. Detailed investigation of secondary substances including photosynthetic pigments was performed for whole lichen thalli but also for axenically cultivated mycobionts and photobionts by methods of UV/VIS-spectrophotometry and two types of high performance liquid chromatography (HPLC). Additionally, a set of chemical tests is presented to confirm the formation of melanic compounds in lichen and mycobiont samples. All investigated lichens reveal various sets of SLCs, except C. gyrosa where only melanin was putatively identified. Such studies will help to assess the contribution of SLCs on lichen extremotolerance, to understand the adaptation of lichens to prevalent abiotic stressors of the respective habitat, and to form a basis for interpreting recent and future astrobiological experiments. As most of the identified SLCs demonstrated a high capacity in absorbing UVR, they may also explain the high resistance of lichens towards non-terrestrial UVR.
After a 15-month exposure on-board the EXPOSE-R2 space platform, situated on the outside of the International Space Station, four astrobiology experiments successfully came back to Earth in March and June 2016. Among them, the BIOMEX (BIOlogy and Mars EXperiment) experiment aims at investigating the endurance of extremophiles and stability of biomolecules under space and Mars-like conditions in the presence of Martian mineral analogues (de Vera et al., 2012). The preservation and evolution of Raman biosignatures under such conditions is of particular interest for guiding future search-for-life missions to Mars (and other planetary objects) carrying Raman spectrometers (such as the Raman Laser Spectrometer instrument on board the future ExoMars rover). The photoprotective carotenoid pigments (present either in photosynthetic organisms such as plants, algae, cyanobac-teria and in some bacteria and archaea) have been classified as high priority targets for biomolecule detection on Mars and therefore used as biosignature models due to their stability and easy identification by Raman spec-troscopy (Böttger et al., 2012). We report here on the first results from the analysis of two carotenoids containing organisms: the cyanobacterium Nostoc sp. (strain CCCryo 231-06; = UTEX EE21 and CCMEE 391) isolated from Antarctica and the green alga cf. Sphaerocystis sp. (strain CCCryo 101-99) isolated from Spitsbergen. Desiccated cells of these organisms were exposed to space and simulated Mars-like conditions in space in the presence of two Martian mineral analogues (phyllosilicatic and sulfatic Mars regolith simulants) and a Lunar regolith analogue and analyzed with a 532nm Raman microscope at 1mW laser power. Carotenoids in both organisms were surprisingly still detectable at relatively high levels after being exposed for 15 months in Low Earth Orbit to UV, cosmic rays, vacuum (or Mars-like atmosphere) and temperatures stresses regardless of the mineral matrix used. Further analyses will help us to correlate these results with survival potential, cellular damages or stability and the different extremophiles tested in the BIOMEX experiment. Böttger, U., de Vera, J.-P., Fritz, J., Weber, I., Hübers, H.-W., and Schulze-Makuch, D. (2012). Optimizing the detection of carotene in cyanobacteria in a martian regolith analogue with a Raman spectrometer for the ExoMars mission. Planetary and Space Science 60, 356–362.
The origin of methane on Mars is unknown. It might originate from geothermal or biological activities. To identify the origin of methane a number of gas detectors are on their way to Mars. Raman spectroscopy is foreseen to be used for life detection. It can be used for detection of methane producing microorganisms (biogenic source of methane). The presentation of a systematically approach for detection of methane producing archaea in a Mars-like environment might give insights in this method.
The GANOVEX X expedition (German Antarcic North Victoria Land Expedition) in the Antarctic summer season 2009/2010 took place on the Antarctic continent. Besides analysis on the geological formations, the distribution of microorganisms as lichens, fungi, green alga and cyanobacteria has been studied along a longitudinal and altitude transect. A diversity of micro-niches has been discovered. Cosmopolites and endemic microorganisms developed adaptation strategies to colonize retreat areas of eroded surfaces, fissures and cracks of granite, volcanic and metamorphic rocks in permafrost regions. These specific habitats were additionally characterized by measurements of the macro- and microclimate (UV-, IR-, VIS-/PAR- radiation, humidity, temperature, atmospheric ozone, water and aerosol content). Based on the discoveries in the mentioned area of the Transantarctic Mountains and due to comparisons to previous results obtained from some of the space-exposed cosmopolites in the space experiments “Lithopanspermia” on BIOPAN 6 / FOTON M3 satellite and “LIFE” on EXPOSE on the International Space Station (ISS) as well as from Mars simulations at the HUMILAB (DLR Berlin), we conclude, that these investigated microorganisms from the Antarctic transect as well as from Spitsbergen and from alpine regions can be characterized as resistant to Mars conditions and that the recent Mars is probably still a habitable planet for Arctic, Antarctic and alpine microorganisms.
Mars is a frozen desert planet. Considerable intense UV radiation fluxes reach its surface and with its thin and 95 % CO2 rich atmosphere and an atmospheric pressure of approximately 6 mbar this planet is not supposed to be habitable. But according to a variety of different experiments during the last decade where microorganisms were investigated under Mars-like environmental conditions there is evidence that even recent Mars appears to be a habitable planet. The habitability of the surface and upper subsurface of Mars depends on one hand on the viability and adaptation capacity of microorganisms under Mars-like environments and on the other hand on the planet's energy resources and liquid water availability. Besides chemical, inorganic energy sources in the soil intense solar radiation is available as additional energy source on the surface of Mars but might be harmful for most of known terrestrial life forms. However, previous studies on extremophilic microorganisms which were performed on space exposure platforms (e.g. BIOPAN on the satellite FOTON and EXPOSE on ISS) show the high resistance of tested bacteria, archaea and lichens to space radiation and desiccation caused by vacuum. During Mars simulation experiments photosynthesizing microorganisms are even able to do photosynthesis periodically. The periodicity of the photosynthetic activity depends on the diurnal cycle with its varying temperatures and relative humidity. It is important to emphasize that the aforementioned space resistant microorganisms are mainly collected in polar and alpine habitats. They are living in permafrost regions with high UV radiation income and extreme dryness provoking high adaptation strategies. Because of these environmental parameters the alpine, desert and polar habitats were characterized as Mars analogue. The Mars analogy of these regions can also be justified by comparing the colonized alpine and polar field profiles with surface structures on Mars. Numerous investigations were done during field campaigns in the Alps, the Arctic (Svalbard) and in Antarctica. Based on these field investigations it becomes obvious that gullies, polygon rich regions and micro caves, fissures and cracks in rocks can be seen as suitable candidates for habitable areas on the surface of Mars in addition to the supposed ice rich environment in the subsurface. As mentioned above, the habitability of Mars depends also on the availability of liquid water. Due to the presence of salts and perchlorate rich soils on Mars water can for sufficient relative humidity remain in a liquid phase, forming at least temporary liquid cryobrines far below the freezing point, which e.g. might be useful for some halophilic microorganisms. This may be in favour of the habitability of the Martian surface. In addition the habitability can also be influenced by the sorption and desorption capacity of other soil particles. These particles and salty solutions could avoid or enhance the liquid phase of water. Processes enhancing the liquefaction of water might explain the recently observed rheological events provoking e.g. the formation of gullies on the surface of Mars which are known as real habitats for a diversity of microorganisms on terrestrial martian analogue environments mostly present in the polar regions. All presently enumerated factors are positively emphasizing that habitability of recent Mars is particularly probable for some of terrestrial life forms.
The search for traces of extinct or extant life in extraterrestrial environments is one of the main goals for astrobiologists; due to their ability to withstand stress producing conditions, extremophiles are perfect candidates for astrobiological studies. The BIOMEX project aims to test the ability of biomolecules and cell components to preserve their stability under space and Mars-like conditions, while at the same time investigating the survival capability of microorganisms. The experiment has been launched into space and is being exposed on the EXPOSE-R2 payload, outside of the International Space Station (ISS) over a time-span of 1.5 years. Along with a number of other extremophilic microorganisms, the Antarctic cryptoendolithic black fungus Cryomyces antarcticus CCFEE 515 has been included in the experiment. Before launch, dried colonies grown on Lunar and Martian regolith analogues were exposed to vacuum, irradiation and temperature cycles in ground based experiments (EVT1 and EVT2). Cultural and molecular tests revealed that the fungus survived on rock analogues under space and simulated Martian conditions, showing only slight ultra-structural and molecular damage.
RLS (Raman Laser Spectrometer - one of the Pasteur Payload Instruments onboard ExoMars 2018) will perform Raman measurements on Mars to identify organic compounds and mineral products as an indication of biological activity. The measurements will be performed on crushed powdered samples inside the Rover's ALD (Analytical Laboratory Drawer). Raman analytics with the same specifications as those onboard the future ExoMars mission are conducted to test their potential of identifying biological material on martian analogue material. Appropriate measurement parameters for the detection of biological material as well as for the determination of the mineral composition will be derived. In addition, we report on problems using Raman spectroscopy to discriminate cells of microorganisms from the mineral background. Two organisms are chosen as test candidates for potential life on Mars: cyanobacteria and methane producing archaea. Prokaryotes like archaea and bacteria appeared on early Earth at least 3.8 to 3.5 billion years ago (Gya). At this time on Mars the climate was more temperate and wet compared to the present day as inferred from geological evidence for liquid water on the ancient martian surface. Thus life might have developed under similar conditions as on Earth or might have been transferred from Earth (or vice versa). Methane is known to be present on Mars, although the origin (if geothermal or biological activity) is still unknown. Cyanobacteria and prokaryotes using photosystem I belong to the oldest microbes on Earth. These organisms use pigments such as scytonemin and β-carotene as UV protection. Especially β-carotene emits a strong Raman signal. Raman analytics are used for detection of biofilm forming cyanobacteria Nostoc commune strain on the below described Mars analogue mineral mixtures. N. commune is known to be resistant to desiccation, UV B radiation and low temperatures, and thus suitable as a candidate for a potential life form on Mars. Furthermore the Raman technique is applied on methane producing archaea candidatus Methanosarcina gelisolum isolated from Siberian permafrost. These archaea are embedded in the martian analogue material for further analysis. Two different Mars analogue materials containing specific mineral mixtures are used in this investigation. The (1) Phyllosilicatic Mars Regolith Simulant (P-MRS) and (2) Sulfatic Mars Regolith Simulant (S-MRS) reflect the current understanding regarding environmental changes on Mars. Weathering or hydrothermal alteration of crustal rocks and of secondary mineralization during part of the Noachian and Hesperian epoch followed by the prevailing cold and dry oxidising condition with formation of anhydrous iron oxides. The use of two different mixtures is based on observations that phyllosilicate deposits do not occur together with sulphate deposits. P-MRS and S-MRS serve as mineral-matrix in which the cells of the microorganisms are embedded. Varying periods of measurement time and number of repetitions are performed to get optimal Raman spectra for cyanobacteria and methanogens. A measurement regime is proposed for mineral mixtures with cyanobacteria on the basis of the RLS instrument characteristics. Raman analytics are capable to identify biosignatures like β-carotene on a multi-mineral mixture similar to those expected to be encountered during the ExoMars mission.
One important mechanism for understanding the interaction between cells and the various radiation sources in space is to perform laboratory analysis on microorganisms or bio-relevant molecules which have been exposed to this radiation environment. However comprehensive scientific assessment of the biological effects caused by exposure to real space environments cannot be simulated in the labs. Many investigation enterprises were performed in the past to approach “deep space exposure experiments” by using simulation facilities in terrestrial laboratories. But the best approach can be reached through exposure on platforms in Low Earth Orbit including satellites like FOTON (with exposure platform BIOPAN) and the International Space Station (with exposure platform EXPOSE-E on the Columbus module, EXPOSE-R on the Zwezda module and exposure platforms on KIBO-module). BIOMEX is such a kind of space exposure experiment to be realized on the International Space Station during the EXPOSE-R2 mission which tries to investigate three different topics as there are (i) the stability and detection of biosignatures or bio-traces in an extraterrestrial environment such as space- and Mars-like conditions, (ii) the limits of life in context to the Lithopanspermia hypothesis by using organisms as test samples of all three domains of the tree of life (archaea, bacteria, eukaryotes) and (iii) to perform tests on the habitability of the planet Mars. Selected results of the environmental verification tests (EVTs) as well as the scientific verification tests (SVT) as part of the research enterprises before exposure to space conditions will be presented. The results clearly show that a big number of organisms might survive space and Mars-like conditions and biosignatures of a set of microorganisms are still characterized by different spectroscopic methods (Raman, IR, UV/VIS). Different applied methods and results obtained by these applications are even of interest for other scientific disciplines as there are archaeology, medicine, engineering technology and space exploration. Thus we can conclude that research in space is of benefit for research on Earth.
In the past decade, various astrobiological studies on different lichen species investigated the impairment of viability and photosynthetic activity by exposure to simulated or real space parameters (as vacuum, polychromatic ultraviolet (UV)-radiation and monochromatic UVC) and consistently found high post-exposure viability as well as low rates of photosynthetic impairment (de Vera et al. 2003, 2004a; 2004b; de la Torre et al. 2010; Onofri et al. 2012; Sánchez et al. 2012, 2014; Brandt et al. 2014). To achieve a better understanding of the basic mechanisms of resistance, the present study subdued isolated and metabolically active photobionts of two astrobiologically relevant lichens to UVC 254 nm , examined its effect on photosynthetic activity by chlorophyll a fluorescence and characterized the UVC-induced damages by quantum yield reduction and measurements of non-photochemical quenching. The results indicate a strong impairment of photosynthetic activity, photoprotective mechanisms and overall photobiont vitality when being irradiated in the isolated and metabolically active state. In conclusion, the present study stresses the higher susceptibility of photobionts towards extreme environmental conditions as UVC-exposure, a stressor that does not occur on the Earth. By comparison with previous studies, the present results highlight the importance of protective mechanisms in lichens, such as morphological–anatomical traits (Meeßen et al. 2013), secondary lichen compounds (Meeßen et al. 2014) and the symbiont's pivotal ability to pass into anhydrobiosis when desiccating.
Samples of the extremotolerant Antarctic endemite lichen Buellia frigida are currently exposed to low-Earth orbit-space and simulated Mars conditions at the Biology and Mars Experiment (BIOMEX), which is part of the ESA mission EXPOSE-R2 on the International Space Station and was launched on 23 July 2014. In preparation for the mission, several preflight tests (Experimental and Scientific Verification Tests, EVT and SVT) assessed the sample preparation and hardware integration procedures as well as the resistance of the candidate organism toward the abiotic stressors experienced under space and Mars conditions. Therefore, we quantified the post-exposure viability with a live/dead staining technique utilizing FUN-1 and confocal laser scanning microscopy (CLSM). In addition, we used scanning electron microscopy (SEM) to investigate putative patterns of morphological-anatomical damage that lichens may suffer under the extreme exposure conditions. The present results demonstrate that Buellia frigida is capable of surviving the conditions tested in EVT and SVT. The mycobiont showed lower average impairment of its viability than the photobiont (viability rates of >83% and >69%, respectively), and the lichen thallus suffered no significant damage in terms of thalline integrity and symbiotic contact. These results will become essential to substantiate and validate the results prospectively obtained from the returning space mission. Moreover, they will help assess the limits and limitations of terrestrial organisms under space and Mars conditions as well as characterize the adaptive traits that confer lichen extremotolerance. Key Words: Astrobiology-BIOMEX-EXPOSE-R2-Extremotolerance-Lichens. Astrobiology 15, xxx-xxx.
Challenges in astrobiology are the realization of space missions to study the habitability of Mars and the icy moons of the Jovian and Saturnian system as well as to investigate potential biosignatures which could be expected on these worlds. Besides Mars the Jovian moon Europa is a promising candidate to be explored. Water driven resurfacing activity of the icy crust proved by the low amount of impact craters on its surface, as well as observations of cryo-volcanos are indicating the presence of liquid water oceans beneath the surface. Fissures and cracks with colored salty deposits coming from the inner side of the supposed global ocean are showing that this ocean could be a habitable environment  and where it would be good to search for signs of life. In addition the Saturnian moon Enceladus is also a promising candidate for the search for life: water plumes are coming out of an ocean which is covered by an ice crust . Some observations made during the Cassini mission also have shown that besides the presence of water and salt a high number of simple and complex organics was observed within these plumes. Because of these observations international and interdisciplinary scientists are working on new types of space missions with the main task to search for life. To realize and support these future space missions, the scientific teams of the ESA-experiments BIOMEX and proposed BIOSIGN are combining for the first time work performed in planetary analog field sites (involving polar and deep sea research) with work in the lab performed in planetary simulation facilities and combined with research in space on specific exposure facilities as there are satellites and the International Space Station (ISS). Studies on biomolecules within these facilities will create a sophisticated back-up data base for astrobiological exploration missions.
The space mission EXPOSE-R2 successfully concluded last February a 15-month exposure of four astrobiology experiments on the outside of the International Space Station. Among them, the BIOMEX (BIOlogy and Mars EXperiment) experiment aims at investigating the endurance of extremophiles and stability of biomolecules under space and Mars-like conditions in the presence of Martian mineral analogues . The preservation and evolution of Raman biosignatures under such conditions is of particular interest for guiding future search-for-life missions to Mars (and other bodies) carrying Raman spectrometers (such as the Raman Laser Spectrometer instrument aboard the future ExoMars rover). Cyanobacterial photoprotective pigments (namely carotenoids) have been classified as high priority targets for biomolecule detection on Mars and therefore used as biosignature models due to their stability and easy identification by Raman spectroscopy . We report here on their preservation potential after ground-based Martian simulations performed in preparation of the BIOMEX experiment and the return of the space exposed samples. As model organisms, cyanobacteria of the genus Chroococcidiopsis were used due to their well-known relevance in astrobiology tasks dealing with the search for life on Mars and future space applications . Chroococcidiopsis cells mixed with two Martian mineral analogues (phyllosilicatic and sulfatic Mars regolith simulants) were exposed to high UV irradiation combined or not with a Martian simulated atmosphere and consecutively analyzed with a 532nm Raman microscope at 1mW laser power (as proposed for the RLS instrument on ExoMars). Carotenoids showed high preservation potentials and were detectable after simulations of the real space exposure mission: 500MJ/m² of full UV (200-400nm) irradiation and Martian simulated atmosphere . Data gathered during the ground-based simulations will contribute to interpret future results from space experiments and guide our search for life on Mars and other bodies of interest. References:  de Vera, J.-P. et al. (2012) Planetary and Space Science 74, 103.  Böttger, U. et al. (2012) Planetary and Space Science 60, 356.  Billi, D. et al. (2013) Advances in Microbiology 03, 80.  Baqué, M. et al. (2015) Origin of Life and Evolution of Biospheres 46, 289.
The space mission EXPOSE-R2 launched on the 24th of July 2014 to the International Space Station is carrying the BIOMEX (BIOlogy and Mars EXperiment) experiment aimed at investigating the endurance of extremophiles and stability of biomolecules under space and Mars-like conditions. In order to prepare the analyses of the returned samples, ground-based simulations were carried out in Planetary and Space Simulation facilities. During the ground-based simulations, Chroococcidiopsis cells mixed with two Martian mineral analogues (phyllosilicatic and sulfatic Mars regolith simulants) were exposed to a Martian simulated atmosphere combined or not with UV irradiation corresponding to the dose received during a 1-year-exposure in low Earth orbit (or half a Martian year on Mars). Cell survival and preservation of potential biomarkers such as photosynthetic and photoprotective pigments or DNA were assessed by colony forming ability assays, confocal laser scanning microscopy, Raman spectroscopy and PCR-based assays. DNA and photoprotective pigments (carotenoids) were detectable after simulations of the space mission (570 MJ/m2 of UV 200–400 nm irradiation and Martian simulated atmosphere), even though signals were attenuated by the treatment. The fluorescence signal from photosynthetic pigments was differently preserved after UV irradiation, depending on the thickness of the samples. UV irradiation caused a high background fluorescence of the Martian mineral analogues, as revealed by Raman spectroscopy. Further investigation will be needed to ensure unambiguous identification and operations of future Mars missions. However, a 3-month exposure to a Martian simulated atmosphere showed no significant damaging effect on the tested cyanobacterial biosignatures, pointing out the relevance of the latter for future investigations after the EXPOSE-R2 mission. Data gathered during the ground-based simulations will contribute to interpret results from space experiments and guide our search for life on Mars.