Search

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

The next two rover missions to Mars, ESA/Roscosmos’s ExoMars2020 and NASA’s Mars2020, will carry for the first time Raman spectrometers potentially able to dectect traces of past or present life. To support and interpret future spectroscopic data correctly a better understanding of potential habitable environments and putative biosignatures, using analogue environments such as the Atacama Desert, is of paramount importance. In the Atacama Desert, one of the driest places on Earth, life has developed adaptive strategies to decreasing amounts of water: from refuges inside or below rocks as endoliths or hypoliths to inside salts in hygroscopic niches (Davila & Schulze-Makuch, 2016). In the hyperarid core one of the last refuges for life are inside salt crusts using deliquescence as a water source or being in the subsurface waiting for transitory episodes of increased moisture (Schulze-Makuch et al., 2018). These adaptive strategies might also apply to putative Martian life which endured a transition from a water rich past to the very harsh surface conditions of the present giving us clues on where to best look for traces of life on the Red Planet. Salt crusts and salt nodules are particularly interesting targets in this regard because they reside on or very near the surface and are thus easily accessible to future robotic missions. In the Atacama, salt nodules have been shown to host photosynthetic organisms containing easily identifiable pigments by Raman spectroscopy such as carotenoids and scytonemin (Vítek et al., 2014). One of the most damaging factors for life and its remains, both in the Atacama and on Mars, is solar radiation. To investigate the spatial distribution of potential Raman signatures in micro-niches we generated georeferenced 3D-reconstructions of the sampling areas using photogrammetry techniques and plotted the dose received according to the nodules’ orientation. We then analysed salt nodule along dry cut north-south thick sections using Raman mapping to infer any relations between the amount of light received and the presence of detectable signal. Preliminary data show an increased presence of carotenoids, scytonemin, and other biomolecules signals on the nodules oriented towards the south, which are the-more-protected sections of the nodules. References Davila, A.F. & Schulze-Makuch, D. (2016) The Last Possible Outposts for Life on Mars. Astrobiology 16:159–168. Schulze-Makuch, D., Wagner, D., Kounaves, S.P., Mangelsdorf, K., Devine, K.G., Vera, J.-P. de, Schmitt-Kopplin, P., Grossart, H.-P., Parro, V., Kaupenjohann, M., Galy, A., Schneider, B., Airo, A., Frösler, J., Davila, A.F., Arens, F.L., Cáceres, L., Cornejo, F.S., Carrizo, D., Dartnell, L., DiRuggiero, J., Flury, M., Ganzert, L., Gessner, M.O., Grathwohl, P., Guan, L., Heinz, J., Hess, M., Keppler, F., Maus, D., McKay, C.P., Meckenstock, R.U., Montgomery, W., Oberlin, E.A., Probst, A.J., Sáenz, J.S., Sattler, T., Schirmack, J., Sephton, M.A., Schloter, M., Uhl, J., Valenzuela, B., Vestergaard, G., Wörmer, L. & Zamorano, P. (2018) Transitory microbial habitat in the hyperarid Atacama Desert. PNAS 115:2670–2675. Vítek, P., Jehlička, J., Ascaso, C., Mašek, V., Gómez-Silva, B., Olivares, H. & Wierzchos, J. (2014) Distribution of scytonemin in endolithic microbial communities from halite crusts in the hyperarid zone of the Atacama Desert, Chile. FEMS Microbiology Ecology 90:351–366.

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 [1] and the BIOMEX [2] 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 [3]. [1] R. Moeller, M. Raguse, S. Leuko, T. Berger, C.E. Hellweg, A. Fujimori, R. Okayasu, and G. Horneck, Astrobiology, 17, 101–109 (2017). [2] 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). [3] 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 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.

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.

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.

After a 15-month exposure onboard 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 [1]. 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, 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 [2]. 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.

Methanogenic archaea are anaerobic chemotrophic microorganisms that meet many of the metabolic and physiological requirements for survival on the martian subsurface. In particular, methanogens from Siberian permafrost are extremely resistant against different types of environmental stresses as well as simulated martian thermo-physical and subsurface conditions, making them promising model organisms for potential life on Mars. Raman spectroscopy is a vibrational spectroscopic technique that has shown a remarkable potential in microbial identification. It provides fingerprint-like information about the overall chemical composition of the samples and allows a nondestructive investigation. The biosignatures of Methanosarcina soligelidi SMA-21 were characterized by CRM during the growth phases at a single-cell level, which presented a high heterogeneity and diversity in the chemical composition of the cells and detectible subpopulation differences. This study also highlighted potential technical challenges concerning the Raman detection of methanogenic archaea (and other non-pigmented microorganisms) embedded on a mineral substrate. The biosignatures of permafrost and non-permafrost strains in the stationary phase of growth were also characterized by CRM. A cluster analysis of the spectra revealed that permafrost and non-permafrost strains have a different overall chemical composition, which has possible evolutionary implications.

BIOMEX (Biology and Mars Experiment) is a space experiment on the exposure platform EXPOSER2 launched by the Progress 56 mission on 24 July and placed on the outer side of the Russian Zvezda Module of the International Space Station (ISS). Twenty-five international institutes are working together and sharing different methods, planetary simulation facilities, and logistics to obtain information about the vitality of the tested microorganisms and the stability of biomolecules as possible biosignatures. This experiment comprises three investigational steps from the field to space: (i) field work with sample collection and habitat characterization at field sites with or without Mars analogy, (ii) Mars simulation experiments in the lab and (iii) exposure to real space conditions. For the second and third steps some of the microorganisms and bio-molecules are embedded in Marsanalog regolith mixtures, placed in compartments enriched with Mars-like CO 2 -atmosphere and exposed to solar irradiation levels approaching those affecting the surface of Mars to test habitability on Mars, as well as the ability to detect the selected, Mars-exposed bio-molecules. One of the aims of this experiment is to investigate the specific bio-related spectra of resistant molecules obtained by fluorescence analysis, Raman-spectroscopy, IR- and UV/VIS spectrometry before and after simulated and real space exposure. The obtained database of stable bio-molecules will support future exploration missions to Mars whose main goal is the search for life.

Even in one of the driest places on Earth, the Atacama Desert, life has found adaptive strategies to decreasing amounts of water: from refuges inside or below rocks as endoliths or hypoliths to inside salts in hygroscopic niches [1]. In the hyperarid core though, one of the last refuges for life are inside salt crusts using deliquescence as a water source or in the subsurface waiting for transitory episodes of increased moisture [2]. These adaptive strategies might also apply to a putative Martian life which endured a transition from a water rich past to the very harsh surface conditions of the present and giving us clues on where to best look for traces of life on the Red Planet. Salt crusts and salt nodules are particularly interesting targets in this regard because they reside on or very near the surface and are thus easily accessible to future robotic missions. In the Atacama, salt nodules have been shown to host photosynthetic organisms containing easily identifiable pigments by Raman spectroscopy such as carotenoids or scytonemin. Mostly composed of halite, they are associated with polygonated soils, but their formation processes are still not fully understood. Salt nodules occur in varying morphologies which can control micro-environmental conditions and possibly microbial colonization (habitation of “micro-niches”). One of the most damaging factors for life and its remains, both in the Atacama and on Mars, is solar radiation. To investigate the distribution of potential Raman signatures in micro-niches we mapped/reconstructed the sampling areas using photogrammetry techniques and plotted the dose received according to the nodules’ orientation. We then analysed salt nodules sections using Raman mapping to infer any relations between the amount of light received and the presence of detectable signal. Raman instruments are indeed part of the next two rover missions to Mars: ESA/Roscosmos’s ExoMars2020 and NASA’s Mars2020. To support and interpret future spectroscopic data correctly, a better understanding of potential habitable environments and putative biosignatures, using analogue environments such as the Atacama Desert, is of paramount importance. [1] A.F. Davila and D. Schulze-Makuch, Astrobiology, 16,159-168, (2016). [2] D. Schulze-Makuch, D. Wagner, S.P. Kounaves, K. Mangelsdorf, K.G. Devine, J.-P. de Vera, P. Schmitt-Kopplin, H.-P. Grossart, V. Parro, M. Kaupenjohann, A. Galy, B. Schneider, A. Airo, J. Frösler, A.F. Davila, F.L. Arens, L. Cáceres, F.S. Cornejo, D. Carrizo, et al., PNAS, 115, 2670–2675 (2018).