Insight of lichens as ideal models for astrobiological studies analyzed by Raman spectroscopy

Exposure experiments of different species to space conditions are essential because real space conditions with different radiation sources like ionizing radiation, UV-radiation, X-rays, gamma-ray from even galactic radiation, vacuum and space weathering by micro-dust cannot simultaneously be simulated in parallel even in our best simulation chambers on Earth. We need results from experiments under real space conditions to enable the development of appropriate predictions about the stability of organisms and their constituent organic parts. The extremophile lichen Circinaria gyrosa is one of the selected species within the BIOMEX (Biology and Mars Experiment) experiment and in this work we compare the previous Raman results obtained in this lichen [1] with the corresponding Raman results on the lichen Xanthoparmelia hueana. Both species have been exposed to space and simulated Mars-like conditions in planetary chambers and we have studied and identified possible degradation process in different layers and biomarkers. The analysis by Raman spectroscopy of simulated Space and Mars exposed samples confirm alterations and damages of the photobiont part of the lichen and changes related to the molecular structure of whewellite. The conclusions of this work will be important to understand what are the effects to consider when biological systems are exposed to space or Mars-like conditions and to expand our knowledge of how life survives in most extreme conditions that is a prerequisite in future planetary exploration projects.Acknowledgment Support for this work was provided by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO), by the project BIOindicadores en MARTE y Espacio (BIOMARSS) (PID2019-109448RB-I00) and by INTA. References [1] M.R. Lopez Ramirez, L.G Sancho, J. P. de Vera, M. Baqué, U. Böttcher, E. Rabbow, J. Martínez-Frías, R. de la Torre Noetzel. Spectrochimica Acta, Part A. 261 (2021) 120046.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Biosignature stability in space enables their use for life detection on Mars

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

Biosignature stability in space enables their use for life detection on Mars

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

Humidity interaction of lichens under astrobiological aspects: the impact of UVC exposure on their water retention properties

We quantitatively studied the hydration and dehydration behaviour of the three astrobiological model lichens Xanthoria elegans, Buellia frigida and Circinaria gyrosa by thermoanalysis and gravimetric isotherm measurements under close-to-Martian environmental conditions in terms of low temperature and low pressure. Additionally, the impact of UVC exposure on the isolated symbionts of B. frigida and X. elegans was studied by thermoanalysis and mass spectrometry as well as by gravimetric isotherm measurements. The thermal analysis revealed whewellite as a component of C. gyrosa which was not found in B. frigida and X. elegans. Neither the water retention nor the thermal behaviour of symbionts changed when irradiated with UVC under dry conditions. On the other hand, UVC irradiation of the wet mycobiont of B. frigida had a distinct impact on the hydration/dehydration ability which was not observed for the mycobiont of X. elegans. Possibly the melanin of B. frigida's mycobiont, that is not present in X. elegans, or a specifically damaged acetamido group of the chitin of B. frigida may be the sources of additional UVC-induced sorption sites for water associated with the UVC exposure

Freeze substitution accelerated via agitation: new prospects for ultrastructural studies of lichen symbionts and their extracellular matrix

2023 Descuento MDPI Slovak Research and Development Agency, contract No. APVV-21-0289 Slovak Grant Agency KEGA, contracts No. 008SPU-4/2023 and No. 009UPJŠ-4/2023 Field campaigns for the collection of C. gyrosa were financed by grants from the Spanish Ministry of Science and InnovationBackground: Lichens, as an important part of the terrestrial ecosystem, attract the attention of various research disciplines. To elucidate their ultrastructure, transmission electron microscopy of resin-embedded samples is indispensable. Since most observations of lichen samples are generated via chemical fixation and processing at room temperature, they lack the rapid immobilization of live processes and are prone to preparation artefacts. To improve their preservation, cryoprocessing was tested in the past, but never widely implemented, not least because of an extremely lengthy protocol. (2) Methods: Here, we introduce an accelerated automated freeze substitution protocol with continuous agitation. Using the example of three lichen species, we demonstrate the preservation of the native state of algal photobionts and mycobionts in association with their extracellular matrix. (3) Results: We bring to attention the extent and the structural variability of the hyphae, the extracellular matrix and numerous crystallized metabolites. Our findings will encourage studies on transformation processes related to the compartmentation of lichen thalli. They include cryopreserved aspects of algal photobionts and observations of putative physiological relevance, such as the arrangement of numerous mitochondria within chloroplast pockets. (4) Conclusions: In summary, we present accelerated freeze substitution as a very useful tool for systematic studies of lichen ultrastructures.Ministry of Education, Science, Research and Sports (Slovak Republic)Ministerio de Ciencia e Innovación (España)Depto. de Farmacología, Farmacognosia y BotánicaFac. de FarmaciaTRUEpubDescuento UC

Freeze Substitution Accelerated via Agitation: New Prospects for Ultrastructural Studies of Lichen Symbionts and Their Extracellular Matrix

(1) Background: Lichens, as an important part of the terrestrial ecosystem, attract the attention of various research disciplines. To elucidate their ultrastructure, transmission electron microscopy of resin-embedded samples is indispensable. Since most observations of lichen samples are generated via chemical fixation and processing at room temperature, they lack the rapid immobilization of live processes and are prone to preparation artefacts. To improve their preservation, cryoprocessing was tested in the past, but never widely implemented, not least because of an extremely lengthy protocol. (2) Methods: Here, we introduce an accelerated automated freeze substitution protocol with continuous agitation. Using the example of three lichen species, we demonstrate the preservation of the native state of algal photobionts and mycobionts in association with their extracellular matrix. (3) Results: We bring to attention the extent and the structural variability of the hyphae, the extracellular matrix and numerous crystallized metabolites. Our findings will encourage studies on transformation processes related to the compartmentation of lichen thalli. They include cryopreserved aspects of algal photobionts and observations of putative physiological relevance, such as the arrangement of numerous mitochondria within chloroplast pockets. (4) Conclusions: In summary, we present accelerated freeze substitution as a very useful tool for systematic studies of lichen ultrastructures

Limits of life at spaceflight conditions: survival of lichens to simulated microgravity

Europlanet Science Congress 2022.Palacio de Congresos de Granada, Spain 18-23 September 2022The search for extraterrestrial life and finding habitable environments on other planets and satellites like Mars, Europa, Enceladus and Titan are a priority of NASA and ESA, since the last decade. To contribute to these highly significant challenges, research has been done with established exposure platforms like those on the Foton satellite and EXPOSE on the ISS expanding now to ESA’s platform Bartolomeo. These were used to expose samples to space vacuum and space radiation, but also to provide gas supply and selected planetary radiation environments. Results obtained by these experiments have allowed to get supplemental knowledge necessary for supporting future investigations to search for life in the universe. Several extremophile lichen species, have been exposed to extraterrestrial environments, i.e. space- and Mars like parameters, during short and long periods on board of ESA’s space missions (Foton M2 and M3, EXPOSE E and R2) to investigate the limits of terrestrial life. To maximize the scientific outcome of these experiments, LICHENS [1], LITHOPANSPERMIA [2], LIFE [3], and BIOMEX [4], a common elaboration and analysis of the results obtained on analogue field studies with results obtained in planetary simulation facilities was necessary to check the survival potential and vitality of the samples before flight. Tests and experiments at different simulation facilities at DLR, and at INTA, included the exposure to space vacuum, space UV radiation and space cosmic radiation, and to Mars-like environment, i.e. Mars atmospheric composition and pressure, as well as Mars UV radiation. Not microgravity or reduced gravity, which is present in space and on Mars, was tried. Here we show the results of the resistance of two extremophile vagrant lichen species, Xanthoparmelia hueana and Circinaria gyrosa, to simulated microgravity (rotation speed of clinostate: 1 rpm) using the UNZIP clinostate at CIB-CSIC (Centro Investigaciones Biológicas Margarita Salas). This is the first time that lichens will be exposed to weightlessness environment in an attempt to isolate the potential contribution of microgravity from other extraterrestrial factors (radiation, vacuum). Combinations of simulated spaceflight conditions, including microgravity, will be necessary to check how this parameter affects the biomolecular level of lichens and their microbiome.Peer reviewe

Cellular Responses of the Lichen Circinaria gyrosa in Mars-Like Conditions

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.The authors acknowledge the Spanish Ministry of Economy, Industry and Competitiveness (MINECO, project SUBLIMAS ESP2015-69810-R). AM and JR thank the MINECO for the Juan de la Cierva–Incorporación (IJCI-2014-20443) and Ramón y Cajal (RYC-2014-16338) post-doctoral contracts, respectively. The authors are grateful to Dr. Ronald Charles Wolf for English improvement. CP and SO thank ASI grant BIOMEX-MCF n. 2013-063-R.0.Peer reviewe