MEXEM – Mars Exposed Extremophile Mixture – a space experiment to investigate the capability of anaerobic organisms to survive on Mars

Assessing the habitability of Mars and detecting life, if it ever existed there, depends on knowledge of whether the combined environmental stresses experienced on Mars are compatible with life as we know it and whether a record of that life could ever be detected. So far, only few investigations were performed to understand the combined effect of different environmental stresses on survival and growth of anaerobic and extremophilic organisms. In the space experiment MEXEM (formerly known as MASE-in-SPACE) the hypothesis will be tested that selected terrestrial organisms, enrichment cultures and original samples from extreme Mars-analogue environments on Earth are able to withstand the Martian environ- mental stress factors due to their highly effective cellular and molecular adaptation and repair mechanisms. In addition, artificially fossilized and aged isolates from Mars-analogue environments on Earth will be examined and assessed with respect to their suitability for biosigna- ture identification. MEXEM samples will be (i) oxygen-depleted natural sediment samples, (ii) natural sediments spiked with selected, defined strains representative for the respective analogue site, (iii) individual (facultative) anaerobic / micro-aerophilic species including ciliates and viruses, (iv) defined mixtures of these biological entities, (v) isolated strains from samples collected inside the ISS and (vi) artificially fossilized isolates from the natural environments. Most of these samples and isolates were obtained from Mars-analogue envi- ronments on Earth in the frame of the EC funded project MASE (Grant Agreement 607297) and from the space experiment EXTREMOPHILES (PI C. Moissl-Eichinger). MEXEM will be flown outside on the ISS in the new exobiology facility ESA is building now. It offers the possibility to simulate of the martian environment, in particular the martian UV cl e, which cannot be done in the lab, but also martian atmosphere and pressure in LEO

MICROORGANISMS FROM MARS ANALOGUE ENVIRONMENTS IN EARTH - COULD THEY SURVIVE ON MARS?

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

BIOMARKERS DETECTION IN MARS ANALOGUE SITES WITHIN 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

Limits of Life and the Habitability of Mars: The ESA Space Experiment BIOMEX on the ISS

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

Mars Analogues for space exploration - from anaerobic field site to culture collection

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 responses of an anaerobic microorganism, Yersinia intermedia MASE-LG-1 to individual and combined simulated Martian stresses

The limits of life of aerobic microorganisms are well understood, but the responses of anaerobic microorganisms to individual and combined extreme stressors are less well known. Motivated by an interest in understanding the survivability of anaerobic microorganisms under Martian conditions, we investigated the responses of a new isolate, Yersinia intermedia MASE-LG-1 to individual and combined stresses associated with the Martian surface. This organism belongs to an adaptable and persistent genus of anaerobic microorganisms found in many environments worldwide. The effects of desiccation, low pressure, ionizing radiation, varying temperature, osmotic pressure, and oxidizing chemical compounds were investigated. The strain showed a high tolerance to desiccation, with a decline of survivability by four orders of magnitude during a storage time of 85 days. Exposure to X-rays resulted in dose-dependent inactivation for exposure up to 600 Gy while applied doses above 750 Gy led to complete inactivation. The effects of the combination of desiccation and irradiation were additive and the survivability was influenced by the order in which they were imposed. Ionizing irradiation and subsequent desiccation was more deleterious than vice versa. By contrast, the presence of perchlorates was not found to significantly affect the survival of the Yersinia strain after ionizing radiation. These data show that the organism has the capacity to survive and grow in physical and chemical stresses, imposed individually or in combination that are associated with Martian environment. Eventually it lost its viability showing that many of the most adaptable anaerobic organisms on Earth would be killed on Mars today

DNA DOUBLE STRAND REPAIR DURING HEAD-DOWN-TILT BEDREST: AGBRESA MEETS RADIATION

BACKGROUND Radiation and reduced gravity impose a major burden on health and performance during human spaceflight. While radiation increases cancer risk and limits tissue regeneration, reduced gravity predisposes to musculoskeletal and cardiovascular deconditioning. Deconditioning could conceivably limit the recovery from radiation damage. Our aim was to develop a terrestrial ex vivo model that could be utilized to study the interaction between simulated reduced gravity using head-down-tilt bed rest and radiation on cellular DNA repair

DNA double strand break repair in peripheral blood mononuclear cells from participants of head-down tilt bedrest studies

Introduction: Major limiting factors in human spaceflight are deleterious effects of reduced gravity and of space radiation exposure on health and performance. Radiationinduced cellular DNA damage, if not repaired or not correctly repaired, increases the risk of cancer and degenerative diseases. Likewise, reduced gravity may lead to musculoskeletal and cardiovascular deconditioning without appropriate countermeasures. We hypothesize that deconditioning could possibly hinder the recovery of cells from radiation damage. We aimed to develop a terrestrial ex vivo model to investigate cellular DNA repair while simulating microgravity effects using head-downtilt (HDT) bedrest during the Spaceflight-Associated Neuro-Ocular Syndrome Countermeasures (SANS-CM) studies. Methods: The SANS CM campaigns comprised three experimental phases: 1) a 14-days baseline data collection (BDC) phase (BDC-14 through BDC-1); 2) 30 days of 6° HDT bedrest phase (HDT1 through HDT30); and 3) a 14- days recovery (R+) phase (R+0 through R+13). Twelve participants were enrolled in each campaign. Blood samples were obtained from the subjects 14 days before the bedrest (BDC-14), 10 and 28 days into head-down-tilt bedrest (HDT-10 and HDT-28) and after 10 days of recovery (R+10). Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Greiner LeucosepTM tubes. We studied the ex vivo induction and repair of DNA double strand breaks, for which the cells were exposed to 1 and 4 Gy of X-rays and harvested after 0.5, 1, 2, 4 and 24 h after irradiation. DNA double strand breaks were detected via immunofluorescence staining of gamma-H2AX that was quantified using flow cytometry. Results: The results for gamma-H2AX fluorescence intensity and the percentage of cells with unrepaired DNA reached a peak value after 2 h of X-rays irradiation and was greatly reduced after 24 h. For all blood collection time-points during the study, BDC-14, HDT-10, HDT-28 and R+10, the gamma-H2AX fluorescence intensity did not differ significantly. Furthermore, there was no significant interpersonal variance of DNA double strand break repair capacity. Conclusion: DNA double strand break repair activity in PBMC remained unaffected by one month of HDT bedrest, suggesting that the physical deconditioning does not modulate DNA repair capacity. Acknowledgement: The study was funded by the National Aeronautics and Space Administration (NASA) and the German Aerospace Center, and performed at the :envihab research facility of the DLR Institute of Aerospace Medicine. The authors wish to gratefully acknowledge the bedrest participants who volunteered their time, without whom this project would not have been possible. We also thank the SANS-CM study staffs for their dedicated work and tireless effort

Mineralization and potential for fossilization of an extremotolerant bacterium isolated from a past Mars analog environement

International audienc

DNA double strand break repair in peripheral blood mononuclear cells from participants of head-down tilt bedrest studies

Introduction: Major limiting factors in human spaceflight are deleterious effects of reduced gravity and of space radiation exposure on health and performance. Radiationinduced cellular DNA damage, if not repaired or not correctly repaired, increases the risk of cancer and degenerative diseases. Likewise, reduced gravity may lead to musculoskeletal and cardiovascular deconditioning without appropriate countermeasures. We hypothesize that deconditioning could possibly hinder the recovery of cells from radiation damage. We aimed to develop a terrestrial ex vivo model to investigate cellular DNA repair while simulating microgravity effects using head-downtilt (HDT) bedrest during the Spaceflight-Associated Neuro-Ocular Syndrome Countermeasures (SANS-CM) studies. Methods: The SANS CM campaigns comprised three experimental phases: 1) a 14-days baseline data collection (BDC) phase (BDC-14 through BDC-1); 2) 30 days of 6° HDT bedrest phase (HDT1 through HDT30); and 3) a 14- days recovery (R+) phase (R+0 through R+13). Twelve participants were enrolled in each campaign. Blood samples were obtained from the subjects 14 days before the bedrest (BDC-14), 10 and 28 days into head-down-tilt bedrest (HDT-10 and HDT-28) and after 10 days of recovery (R+10). Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Greiner LeucosepTM tubes. We studied the ex vivo induction and repair of DNA double strand breaks, for which the cells were exposed to 1 and 4 Gy of X-rays and harvested after 0.5, 1, 2, 4 and 24 h after irradiation. DNA double strand breaks were detected via immunofluorescence staining of gamma-H2AX that was quantified using flow cytometry. Results: The results for gamma-H2AX fluorescence intensity and the percentage of cells with unrepaired DNA reached a peak value after 2 h of X-rays irradiation and was greatly reduced after 24 h. For all blood collection time-points during the study, BDC-14, HDT-10, HDT-28 and R+10, the gamma-H2AX fluorescence intensity did not differ significantly. Furthermore, there was no significant interpersonal variance of DNA double strand break repair capacity. Conclusion: DNA double strand break repair activity in PBMC remained unaffected by one month of HDT bedrest, suggesting that the physical deconditioning does not modulate DNA repair capacity. Acknowledgement: The study was funded by the National Aeronautics and Space Administration (NASA) and the German Aerospace Center, and performed at the :envihab research facility of the DLR Institute of Aerospace Medicine. The authors wish to gratefully acknowledge the bedrest participants who volunteered their time, without whom this project would not have been possible. We also thank the SANS-CM study staffs for their dedicated work and tireless effort