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The identification of reliable biomarkers, such as amino acids, is key for the search of extraterrestrial life. A large number of microorganisms metabolize, synthesize, take up and excrete amino acids as part of the amino acid metabolism during aerobic and/or anaerobic respiration or in fermentation. In this work, we investigated whether the anaerobic microbial metabolism of amino acids could leave a secondary biosignature indicating biological activity in the environment around the cells. The observed fingerprints would reflect the physiological capabilities of the specific microbial community under investigation. The metabolic processing of an amino acid mixture by two distinct anaerobic microbial communities collected from Islinger Mühlbach (ISM) and Sippenauer Moor (SM), Germany was examined. The amino acid mixture contained L-alanine, β-alanine, L-aspartic acid, DL-proline, L-leucine, L-valine, glycine, L-phenylalanine and L-isoleucine. In parallel, an amino acid spiked medium without microorganisms was used as a control to determine abiotic changes over time. Liquid chromatography mass spectrometry (LC-MS) was used to track amino acid changes over time. When comparing to the control samples that did not show significant changes of amino acids concentrations over time, we found that glycine was almost completely depleted from both microbial samples to less than 3% after the first two weeks- This results indicates a preferential use of this simple amino acid by these microbial communities. Although glycine degradation can be caused by abiotic processes, these results show that its preferential depletion in an environment would be consistent with the presence of life. We found changes in most other amino acids that varied between amino acids and communities, suggesting complex dynamics with no clear universal pattern that might be used as a signature of life. However, marked increases in amino acids, caused by cellular synthesis and release into the extracellular environment (e.g., alanine), were observed and could be considered a signature of metabolic activity. We conclude, that substantial anomalous enhancements of some amino acids against the expected abiotic background concentration may be an agnostic signature of the presence of biological processes.

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 Tech

Plant cultivation in large-scale closed environments is challenging and several key technologies necessary for space-based plant production are not yet space-qualified or remain in early stages of development. The EDEN ISS project (EC Horizon 2020 RIA, grant no. 636501, https://eden-iss.net/) developed and demonstrated higher plant cultivation technologies, suitable for future deployment on the International Space Station and from a long-term perspective, within Moon and Mars habitats. The cultivation of fresh vegetables in the EDEN-ISS greenhouse was tested for nine months in Antarctica. Throughout the cultivation period the microbial bioburden and biodiversity was monitored inside the greenhouse on a regular basis to assess the potential microbial risks associated with plant and human pathogens. Samples were taken from plants, surfaces inside the greenhouse, plant nutrients solutions and the fresh water tank. From preliminary results of the ongoing analysis, it can be stated that in general, the quantity of microorganisms, i.e. bacteria, molds and yeasts, was fluctuating substantailly during the nine months. The microbial load in the nutrition solution as well as the surface contamination increases over time. Almost all microorganisms found on the plant samples are molds. The quantity as well as the diversity of microorganisms on the plant surfaces is very low compared to commercially produced vegetables from a German grocery.

The cold, arid, remotely located and perennially ice covered environment of the Antarctic ice sheet is the most hostile place on Earth. It has long been considered an analogue to how life might persist in the frozen landscape of the major Astrobiological targets of our solar system such as Mars or the Jupiter’s ice-covered moon Europa. In the frame of the ICExPOSE project presented here, the parameters outside the Antarctic Concordia station are utilized as a testbed for performed or planned long-duration space flights and to study the survivability of selected test organisms in an extremely cold (with temperature swings) and highly variable UV environment. The most likely terrestrial organisms to endure such an excursion are extremely tolerant and/or (multi-) resistant microbes-extremophiles- that have evolved mechanisms to withstand such severe conditions. The survivability of a variety of human-, space-flight and extreme-associated microorganisms from all three domains of life (plus viruses) will be investigated using a multiuser exposure facility called EXPOSE that has already been successfully flown on ISS for space exposure durations of up to 2 years. The EXPOSE Mission Ground Reference (MGR) trays are still available and will be reused to accommodate the samples for passive exposure. Microbiological response to single and combined extraterrestrial conditions including simulations of astrobiological relevant environments, like simulated Martian atmospheric conditions, will be tested. The scientific questions addressed in ICEXPOSE are: how is the survival of human-associated and Polar Regions- derived microorganisms compared to (other) environmental extremophilic microorganisms; which physiological state (i.e., cells, spores or colony/biofilms) harbors the weakest or strongest viability and/or mutagenicity detectable after exposure; what type of morphologic and molecular changes can be identified and to which extent does the exposure conditions (e.g. UV-exposed versus UV-shielded) influence the microbial physiology (e.g. pathogenicity, antibiotic resistance, and metabolism) of the exposed species. The results of the ICExPOSE experiment will provide valuable information on: the definition of the physical-chemical limits of life as well as the potential habitability of other planetary bodies; the assessment of the risk of microbial contamination inside human inhabited confined areas and consequent challenges for human health; how to better monitor and control microbial contamination in spaceflight environments, as a key-factor for the success of future space exploration missions; whether specific microorganisms pose possible forward contamination risks that could impact planetary protection policy and will provide complementary results for the two selected future ESA space experiments MEXEM and IceCold.

Exploration of the solar system, needs science and technology support to work at a merged way. Space platforms, such as EXPOSE, are a priority for the performance of experiments which are focused on the exploration of the limits of terrestrial life, trying to get responses to some questions, such as the 1) survival capacity of biological organisms in space, 2) the effects of space environment on no protected biological and chemical material and 3) results of degradation of organic molecules (biomarkers), that will support the interpretation of missions, such as Rosetta, ExoMars and Curiosity). Some priority scientific objectives for the next 10 years are focused on "Life and habitability" and "Biomarkers for detection of extraterrestrial life", what is belonging to the main aims of BIOMEX (Biology- and Mars Experiment, 2014-2016, ESA) [1,3], experiment performed on the exposure platform EXPOSE-R2 on board of ISS.

Exploration of the solar system is a priority research area of the AstRoMap European Astrobiology Roadmap (Horneck et al., 2015) [1], focused on various research topics, one of them is “Life and Habitability” and an other one is “Biomarkers for easy the detection of life”. Therefore “space platforms and laboratories” are necessary, such as EXPOSE, to gain more knowledment of space- and extraterrestrial habitats, eventually for human interplanetary exploration (Space, Moon, Mars, Encedalus, Titan, Europa). At the exposure platform EXPOSE-R2 on ISS (2014-2016), samples of the astrobiological model system Circinaria gyrosa gyrosa [3,4,5,6], belonging to the BIOMEX experiment [2], (Biology and Mars Experiment, ESA), were exposed during 18 months to space and to a Mars simulated environment, to study Mars habitability and resistance to space conditions. The data obtained by the investigation on biomarkers after being exposed to Mars-like conditions will support the analysis of data obtained during future instrumental detection operations in future space missions on Mars (i.e. ExoMars). After the return of the samples in June 2016, the first preliminary analysis showed a quick and complete recovery of metabolic activity of the control samples exposed to space vacuum and Mars-like atmosphere. In contrast, the samples directly exposed to extraterrestrial UV solar radiation showed slow recovery, in reference to their observed original activity. Here we expose the last results that show the biomolecular changes of the DNA analized by PCR and complementary sequencing techniques, in correlation with the previous results supporting changes in metabolic activity, and changes in viability (Electron- and fluorescence microscopy techniques), as well as in morphology/ultrastructure due to space vacuum and Mars atmosphere. Additionaly, the biogeochemical variations have been 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. Moreover, differences were observed between samples irradiated with extraterrestrial UV solar radiation and samples positioned below defined as dark-control samples. These experiments will contribute to answer questions on the habitability of Mars, on the likelihood of the Lithopanspermia Hypothesis and will be of relevance for planetary protection issues.

Exploration of the solar system is a priority research area of the AstRoMap European Astrobiology Roadmap, focusing on several research topics, such as “Life and Habitability” andan other one is “Biomarkers for easy the detection of life”. Therefore, “space platforms and laboratories”, as the EXPOSE setup installed outside the ISS, are essential to gain more knowledge of spaceand planetary environments, which might be an essential basis for improvement of the robotic and human interplanetary exploration. At the exposure platform EXPOSE-R2 on the ISS (2014-2016), samples of the astrobiological model lichen Circinaria gyrosa, a specie which was exposed 18 months to space and simulated Mars-like conditions during the BIOMEX experiment (Biology and Mars Experiment, ESA), was investigated, to study Mars’ habitability and resistance to space conditions. The data obtained by this biomarker-study after being exposed to Marslike conditions will support the analysis of data obtained during future instrumental detection operations in future space missions on Mars (i.e. ExoMars or Mars 2020). N/A

International audience; 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 paper reviews 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.

Introduction: The Mars Analogues for Space Ex-ploration (MASE) project is brining new insight about Mars potential habitability but also some new knowledge about Earth organisms and the functioning of extreme terrestrial ecosystems. The overall aim of the MASE project is to study a variety of Mars-like environments in order to further our understanding of Martian habitability, as well as our ability to detect organisms that might be present on Mars. This collabo-rative, 4 research project supported by the European Commission’s Seventh Framework Programme has been running since January 2014 and a variety of dis-semination and communication activities has been per-formed since then to increase visibility of Mars ana-logues research.

International audience; The artificial mineralization of a polyresistant bacterial strain isolated from an acidic, oligotrophic lake was carried out to better understand microbial (i) early mineralization and (ii) potential for further fossilisation. Mineralization was conducted in mineral matrixes commonly found on Mars and Early-Earth, silica and gypsum, for 6 months. Samples were analyzed using microbiological (survival rates), morphological (electron microscopy), biochemical (GC-MS, Microarray immunoassay, Rock-Eval) and spectroscopic (EDX, FTIR, RAMAN spectroscopy) methods. We also investigated the impact of physiological status on mineralization and long-term fossilisation by exposing cells or not to Mars-related stresses (desiccation and radiation). Bacterial populations remained viable after 6 months although the kinetics of mineralization and cell-mineral interactions depended on the nature of minerals. Detection of biosignatures strongly depended on analytical methods, successful with FTIR and EDX but not with RAMAN and immunoassays. Neither influence of stress exposure, nor qualitative and quantitative changes of detected molecules were observed as a function of mineralization time and matrix. Rock-Eval analysis suggests that potential for preservation on geological times may be possible only with moderate diagenetic and metamorphic conditions. The implications of our results for microfossil preservation in the geological record of Earth as well as on Mars are discussed. Redrawing the history of early life on Earth requires being able to assess if microstructures present in the oldest terrestrial rocks are of biological origin or not. Such assessments are still very challenging mainly due to the degradation of microbial remains during diagenesis and to microbial-like morphologies abiotically produced. Several Archaean rocks could nevertheless be described as ancient unambiguous biological systems, representative of the early-Earth fossil record, like strata of South Africa and Australia containing evidence of phototrophic 1–6 and heterotrophic microbial. To better understand the processes leading to microfossil formation and preservation, artificial mineralization of microorganisms, also called fossilisation, was first undertaken by Oehler and Schopf with the silicification of

Assessing the habitability of Mars and detecting life, if it was ever there, depends on knowledge of whether the combined environmental stresses experienced on Mars are compatible with life and whether a record of that life could ever be detected. Many combinations of Mars relevant stress factors, such as high radiation dose rates and high UV uences combined with high salt concentrations, and low water activity, have not been investigated. In particular, the response of anaerobic organisms to Mars-like stress factors and combinations thereof are not known. In the EC project MASE (Mars Analogues for Space Exploration) we address these limitations by characterising different Mars analogue environments on Earth, isolating microorganisms from these sites and exposing them to Mars relevant stress factors alone and in combination. We want to find out, if these bacteria respond in an additive or synergistic way and if they would be able to survive on Mars. So far, eight only distantly related microorganisms are under detailed investigation, e.g Yersinia sp., Halanaerobium sp., Acidiphilum sp. Desulfovibrio sp.. Unexpectedly, a Yersinia strain turned out to be quite resistant, especially against desicca- tion and oxidising compounds, whereas a Desulfovibrio sp. strain exhibit a relatively high radiation resistance. The future experiments aim at the identification of the underlying cellu- lar and molecular mechanisms and the comparison to other new isolates from Mars analogue environments on Earth in the MASE project.

Life is a physico-chemical process by which tell-tale signals or traces are left on the environment. These signals are indicators of life and are known as biomarkers. Besides, the traces of some kinds of microorganisms can be well preserved, provided that they are rapidly mineralized and that the sediments in which they occur are rapidly cemented [1]. The search for these traces of life is one of the main objectives of Mars exploration [1] and to improve and optimize the search and detection of them forms part of MASE project targets. In MASE project (Mars Analogues for Space Exploration) we work to improve approaches and methods for biomarker detection in samples with low biomass from Mars analogue sites. A developed antibody multiarray competitive immunoassay (MACIA) for the simultaneous detection of compounds of a wide range of molecular sizes or whole spores and cells [2] [3] has revealed as suitable option to achieve this purpose.

The MASE (Mars Analogues for Space Exploration) [1] project is a four-year collaborative research project supported by the European Commission Seventh Framework Contract. The aim of the project is to understand how combined environmental stresses influence the habitability of a number of Mars analogue environments in Europe and beyond. Field sites sampled for MASE include deep subsurface salts at Boulby Mine in the UK, sulfidic springs In Germany and an acidic cold lake in lceland. Samples and isolates have also been provided to the project from acidic deep subsurface environments at the Rio Tinto in Spain as well as permafrost sites in Russian Siberia and northern Canada. Crucial to assessing the habitability of any environmental system to be used as an astrobiological analogue • whether for life in general or, as in this case, for anaerobic microorganisms • is a detailed understanding of their geological and physiochemical context [2, 3]. One of the key outcomes of the MASE project is a comparison and synthesis of just such a set of context data from a varied set of analogue sites, the core of which is presented here, and complemented by an analysis of field samples to detect and quantify amino acids, other organics as well as biologically relevant molecules. We show that anaerobic environments provide some of the best fidelity environments for examining the potential habitability of environments on Mars, which are also anaerobic, but that even these analogue environments show the signatures of Earth's very different geological history, such as high carbon abundance in some environments (from 0.1% in lcelandic lake sediments to 22. 7% in deep .permafrost environments).

Astrobiology seeks to understand the limits of life and to determine the physiology of organisms in order to be able to better assess the potential habitability of other worlds and improve our ability to assay them for the presence of life. To successfully achieve this we require representative microorganisms from environments on Earth that in physical and/or chemical conditions approximate to extraterrestrial environments. The most challenging of these environments with respect to the sample collection and follow on isolation and cultivation of microorganisms are anaerobic environments. Here we describe a systematic approach to this challenge and aim to provide a guideline for future fieldwork and sampling campaigns. We selected a number of anaerobic environments based on characteristics that make them analogous to past and present locations on Mars (Icelandic lakes, sulfidic springs, deep hypersaline environments, acidic iron-rich environments, and permafrost). We implemented a culturing approach to enrich organisms from these environments under anaerobic conditions using a defined medium that would allow for all organisms to be grown under identical culturing conditions m future physiological comparisons. We then isolated anaerobic microorganisms, carried out a study of their basic physiology and deposited these organisms in the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) culture collection to make them available to astrobiologists and microbiologists. This project represents the first attempt to implement a coordinated effort from the selection of extraterrestrial analog sites through to the isolation and the characterisation of organisms and their deposition within a culture collection.

The search for life beyond Earth is challenging and requires, as a prerequisite, intensive research on microbial life in similar, extreme environments on Earth. Mars analogue sites are characterised by e.g. anoxic conditions, organic compound limitation, low temperatures, high salinity or presence of oxidising compounds, and consequently represent the chemical and physical borders of life as we know it. The analysis of microorganisms withstanding such conditions is embedded in the European Commission-funded MASE (Mars Analogues for Space Exploration; (http://mase.esf.org/) project. Combining a broad spectrum of interdisciplinary expertise, the European project members aim at a better understanding of habitability, microbial lifestyles and biomarker preservation in Mars analogues. For the first time, the selected sites (e.g. salt mine, sulfidic springs) have undergone a profound analysis of their microbial communities on various levels, including vast cultivation of anaerobic microorganisms and molecular screening. In this work, we applied propidium monoazide in order to distinguish between cells with intact membrane (considered as viable) and dead cells on molecular stage, followed by DNA extraction, and amplicon-sequencing of the archaeal and bacterial 16S rRNA genes. The geochemistry of the sites was comprehensively investigated (i.e. elemental analysis, amino acid chirality, minerology), to determine triggers for microbial community composition. We aim to set up a model of potential metabolism reactions based on the different setting conditions and compare it with microbiome data. Consequently, we will obtain insights into the prerequisites of possible extra-terrestrial life forms and into their lifestyles, which may enable them to thrive under most extreme conditions.

Introduction: The Life Detection System (LDS) is designed as a two modules system for microbial life detection under growing conditions. The microbes growth is followed by redox, pH and conductivity parameters but others parameters can be monitored as well if needed. The experiments presented in this paper follow the physicochemical parameter in a growth culture under fossilization/mineralization-induced process with the objectives of biomarkers detection. The study of biomarkers detection [1] and fossilization process is crucial from an astrobiological point of view for the search for life on Mars as it has been reported that life can survive on Mars surface conditions under protected microniches [2]. At the same time, and using LDS system, we can follow the modification of some parameters on the media that could drive the process.

MASE (Mars Analogues for Space Exploration) is a four year collaborative research project supported by an EC FP7 contract. Its aim is to understand how combined environmental stresses affect the habitability of a number of Mars analogue environments on Earth, specifically for anaerobic organisms. Crucial to assessing the habitability of any environmental system is a detailed understanding of the geological, physiochemical and biological context in which the environment is set. One of the key outcomes of MASE is a comparison and synthesis of just such a collection of context data from a varied set of Mars analogue sites.

Shock recovery experiments were performed with an explosive set-up in which three types of microorganisms embedded in various types of host rocks were exposed to strong shock waves with pressure pulse lengths of lower than 0.5s: spores of the bacterium Bacillus subtilis, Xanthoria elegans lichens, and cells of the cyanobacterium Chroococcidiopsis sp. 029. In these experiments, three fundamental parameters were systematically varied (1) shock pressures ranging from 5 to 50GPa, (2) preshock ambient temperature of 293, 233 and 193K, and (3) the type of host rock, including nonporous igneous rocks (gabbro and dunite as analogs for the Martian shergottites and chassignites, respectively), porous sandstone, rock salt (halite), and a clay-rich mineral mixture as porous analogs for dry and water-saturated Martian regolith. The results show that the three parameters have a strong influence on the survival rates of the microorganisms. The most favorable conditions for the impact ejection from Mars for microorganisms would be (1) low porosity host rocks, (2) pressures

Petrographic and biological analysis of shock recovery experiments confirms the possible life transport due to an impact from Mars to Earth.