GEOCHEMICAL MARS ANALOG. C. Escudero 1,2 , N. Rodriguez 1,2 , R.Amils 1,2 , D. A. Carrizo 1 , E. R. Uceda 2 , A. G. Fairén 1,3 , F. Gómez 1 and J. L. Sanz 4 . 1 Departamento de Planetología and Habitabilidad, Centro de Astrobiología (INTA-CSIC) Ctra. Ajalvir km 4 Torrejón de Ardoz, Madrid, Spain, 2 Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Madrid, Spain, 3 Department of Astronomy, Cornell University, Ithaca, NY, USA. 4 Departamento de Biologia Molecular, Universidad Autónoma de Madrid, Madrid, Spain. (cescudero@cab.inta-csic.es)

The study of plants adapted to an extreme environment with a high concentration of iron such as Río Tinto allowed the study of important elements for the development and control of plant growth including their localization, management, and storage. The absorption, transport, and accumulation of iron were studied in different species of dicotyledons (Sarcocornia pruinosa, Salicornia patula, Arthrocnemum macrostachyum, and Halogeton sativus of the Chenopodiaceae family) and monocotyledons (Imperata cylindrica, Cynodon dactylon, and Panicum repens from the Poaceae family), all obtained from the Río Tinto banks in different sample collection campaigns. The results clearly show that phytoferritin is not observed in the chloroplast of monocotyledons, an important difference from what is observed in dicotyledons. The presence of plastids with a high concentration of iron in the sieve tubes of monocotyledons strongly suggests their possible role in the transport and accumulation of iron in these plants.

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.

Our understanding of the habitability of Mars is hampered by a lack of knowledge of the stress respons-es of anaerobic organisms. In the project MASE (Mars Analogues for Space Exploration) representative (fac-ultative) anaerobic microorganisms are isolated from Mars analogue environments on Earth and exposed to stresses typical for the Martian environment. The aim is to gain fundamental insights into the limits of anaer-obic microbial life on Earth and to use these data to assess the habitability of Mars.

During the project MASE (Mars Analogues for Space Exploration) several microbial isolates were obtained and some of these strains were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions; e.g. radiation, low water activity, high salt concentrations, oxidizing compounds. All sampling, isolation, and cultivation steps, as well as the stress tests were performed under anoxic conditions. So far, five only distantly related microorganisms are under detailed investigation: \textit{Buttiauxella} sp. MASE-IM-7, \textit{Clostridium} sp. MASE-IM-4, \textit{Halanaerobium} sp. MASE-BB-1, \textit{Trichococcus} sp. MASE-IM-5, and \textit{Yersinia} sp. MASE-LG-1. It was shown that tolerance to desiccation and to ionizing radiation, applied separately was not correlated. If desiccation and exposure to radiation was applied together typical additive effects could be observed in each species. The survival after addition of oxidizing compounds (hydrogen peroxide; perchlorates) for a dedicated time (15 minutes; 24 hours) was very divers. If the five microorganisms were compared with each other, \textit{Trichococcus} sp. MASE-IM-5 was the most sensitive strain and survived only 10 mM hydrogen peroxide for 15 minutes and 24 hours, respectively. The most tolerant organism was \textit{Halanaerobium} sp. MASE-BB-1 which was able to survive 100 mM hydrogen peroxide for both tested time points. In between, the other Bacteria were arranged with different tolerances against hydrogen peroxide. Comparable, species specific results were obtained after the addition of different perchlorates. In general, the strategy of the MASE project has proven to be useful to gain new model microorganisms. The isolated and characterized MASE strains have so far unknown high tolerances against cell damaging treatments and may serve as model organisms for future space exposure experiments.

Introduction: Assessing the biological origin of microstructures present in rocky materials is very chal-lenging mainly due to the degradation of microbial remains during diagenesis and to mineralogical processes leading to abiotic artifacts. To better understand the processes leading to microfossil formation and preservation, artificial mineralisation of microorganisms was first undertaken with the silicification of Cyanobacteria [1], followed by the mineralisation of eukaryotic and bacterial models [2] or of natural communities [3] and has since been notably extended to hyperthermophilic Archaea [4].

During the development of the Mars Analogues for Space Exploration project (MASE) several microbial isolates of the Tessaracoccus genus, a denitrifying actynomicetes, were obtained from enrichment cultures of samples from cores drilled at different depths in the Iberian Pyrite Belt. Some of these strains, isolates SSRT5 and SSRT8, were subjected to Mars environmental stress tests under controlled laboratory conditions; e.g. high salt concentra- tions, temperature, oxidizing compounds, radiation and low water activity. All sampling, isolation, and cultivation steps, as well as the stress tests were performed under strict anoxic conditions. The differences found between these isolates are remarkable. Isolate SSRT8 was able to grow efficiently at low temperature (4 oC). It was able to grow in the presence of 300 mM Ca, Mg and Na perchlorates and to resist up to 1M concentration of these salts. The sensitivity of isolate SSRT5 was dependent on the cation of the perchlorate. Isolate SSRT8 was more resistant to extended exposures to low temperatures (-80 oC) and exposure to 500Gy X-Ray radiation. Both isolates are extremely sensitive to freeze and thaw cycles, desiccation and starvation. Genomic comparison of both isolates is currently underway to detect the een two isolates from the same ecosystem.

Osmotic stress is one of the major limitations for cell growth. Microorganisms can evolve adaptations to abiotic stresses like high salt concentrations in the environment. Sensor and signal transduction networks provide information to the cell about the osmolarity of its surroundings leading to an immediate metabolic response to counteract the osmotic stress. Some of these adaptations can be structural, some are metabolic. Our current knowledge of microbial responses to osmotic challenges is based on studies of representative bacteria, archaea, and eukaryotic microbes that is mainly focusing on the effect of NaCl. There is still a lack of understanding whether and how different salts, for example NaCl versus MgSO4 alter the response of a microorganism to salt induced stress conditions. We chose Yersinia intermedia MASE-LG1 a strain isolated from an Icelandic lake as test organism. It is known for its abilities to adapt to a wide variety of habitats of rapidly changing environmental conditions. In order to identify which roles the different salts play in the global metabolic response, Y. intermedia was exposed sustained salt stress induced by either MgSO4 or NaCl. After metabolite extraction, metabolic profiles from three replicate cultures of Y. intermedia MASE-LG-1 grown under 3 different conditions (e.g. control salt stressed in MgSO4, and salt stressed in NaCl) were obtained. Generally, changes in numerous metabolites mainly in the amino acid metabolisms were observed in stressed samples compared to the control. To a lesser extent the carbohydrate metabolism was also affected. Looking at the effect of the different kations, the results clearly indicated significant differences in response to salt stress induced by the magnesium salt compared to sodium chloride. The results suggest that the amino acid synthesis, reflecting the general activity of translation operations, dominates the reaction to osmotic stress. These adaptations might provide necessary energy and building blocks to fuel processes conveying salt tolerance like the biosynthesis of compatible solutes. In addition we were able to identify metabolites which are linked to osmoprotective activity. The outcome of this study will have impact on our understanding of how microorganisms adapt to hostile environmental conditions.

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.

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.

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.

During the project MASE (Mars Analogues for Space Exploration) several isolates were obtained and these strains were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions; e.g. radiation, low water activity, high salt concentrations, oxidizing compounds. First screening steps were always the microorganisms’ tolerance to survive times of desiccation and exposure to ionizing radiation. Both treatments were performed under anoxic conditions.

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.

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.

This study presents a comprehensive revision of the genus Sarcocornia (Chenopodiaceae) on the Iberian Peninsula based on macromorphological, micromorphological and phylogenetic data, and considering caryological, ecological and biogeographical information. Three species of Sarcocornia have been identified on the Iberian Peninsula: Sarcocornia perennis (Miller) A.J. Scott, Sarcocornia fruticosa (L.) A.J. Scott and Sarcocorniaalpini (Lag.) Rivas-Martínez. Several authors have proposed that S.alpini is a specific and subspecific rank of S. perennis. Fuente, Rufo and Sánchez-Mata have recently described a new species, Sarcocornia hispanica. The micromorphological and molecular studies (sequence of the internal transcribed spacer region) indicate that there is a broad diversity within Sarcocornia in the Western Mediterranean. This article proposes a new species (Sarcocornia pruinosa) and subspecies (S. alpini subsp. carinata) in view of the new data.

Here we describe biogeochemical processes that lead to the generation of acid rock drainage (ARD) and rock weathering on the Antarctic landmass and describe why they are important sources of iron into the Antarctic Ocean. During three expeditions, 2009-2011, we examined three sites on the South Shetland Islands in Antarctica. Two of them displayed intensive sulfide mineralization and generated acidic (pH 3.2-4.5), iron-rich drainage waters (up to 1.78 mM Fe), which infiltrated as groundwater (as Fe(2+)) and as superficial runoff (as Fe(3+)) into the sea, the latter with the formation of schwertmannite in the sea-ice. The formation of ARD in the Antarctic was catalyzed by acid mine drainage microorganisms found in cold climates, including Acidithiobacillus ferrivorans and Thiobacillus plumbophilus. The dissolved iron (DFe) flux from rock weathering (nonmineralized control site) was calculated to be 0.45 × 10(9) g DFe yr(-1) for the nowadays 5468 km of ice-free Antarctic rock coastline which is of the same order of magnitude as glacial or aeolian input to the Southern Ocean. Additionally, the two ARD sites alone liberate 0.026 and 0.057 × 10(9) g DFe yr(-1) as point sources to the sea. The increased iron input correlates with increased phytoplankton production close to the source. This might even be enhanced in the future by a global warming scenario, and could be a process counterbalancing global warming.

The Iberian Pyrite Belt (IPB, southwest of Spain), the largest known massive sulfide deposit, fuels a rich chemolithotrophic microbial community in the Río Tinto area. However, the geomicrobiology of its deep subsurface is still unexplored. Herein, we report on the geochemistry and prokaryotic diversity in the subsurface (down to a depth of 166 m) of the Iberian Pyritic belt using an array of geochemical and complementary molecular ecology techniques. Using an antibody microarray, we detected polymeric biomarkers (lipoteichoic acids and peptidoglycan) from Gram-positive bacteria throughout the borehole. DNA microarray hybridization confirmed the presence of members of methane oxidizers, sulfate-reducers, metal and sulfur oxidizers, and methanogenic Euryarchaeota. DNA sequences from denitrifying and hydrogenotrophic bacteria were also identified. FISH hybridization revealed live bacterial clusters associated with microniches on mineral surfaces. These results, together with measures of the geochemical parameters in the borehole, allowed us to create a preliminary scheme of the biogeochemical processes that could be operating in the deep subsurface of the Iberian Pyrite Belt, including microbial metabolisms such as sulfate reduction, methanogenesis and anaerobic methane oxidation.

Oxidative stress, high radiation doses, low temperature and pressure are parameters which made Mars's surface adverse for life. Those conditions found on Mars surface are harsh conditions for life to deal with. Life, as we know it on Earth, needs several requirements for its establishment but, the only “sine qua nom” element is water. Extremophilic microorganisms widened the window of possibilities for life to develop in the universe, and as a consequence on Mars. Recently reported results in extreme environments indicate the possibility of presence of “oasys” for life in microniches due to water deliquescence in salts deposits. The compilation of data produced by the ongoing missions (Mars Global Surveyor, Mars Odyssey, Mars Express and Mars Exploration Rover Opportunity) offers a completely different view from that reported by Viking missions: signs of an early wet Mars and rather recent volcanic activity. The discovery of important accumulations of sulfates, and the existence of iron minerals like jarosite, goethite and hematite in rocks of sedimentary origin has allowed specific terrestrial models related with this type of mineralogy to come into focus. Río Tinto (Southwestern Spain, Iberian Pyritic Belt) is an extreme acidic environment, product of the chemolithotrophic activity of microorganisms that thrive in the massive pyrite-rich deposits of the Iberian Pyritic Belt. The high concentration of ferric iron and sulfates, products of the metabolism of pyrite, generate a collection of minerals, mainly gypsum, jarosite, goethite and hematites, all of which have been detected in different regions of Mars. Some particular protective environments or elements could house organic molecules or the first bacterial life forms on Mars surface. Terrestrial analogs could help us to afford its comprehension.We are reporting here some preliminary studies about endolithic niches inside salt deposits used by phototrophs for taking advantage of sheltering particular light wavelengths. These acidic salts deposits located in Río Tinto shelter life forms which are difficult to localize by eye. Techniques for its localization and study during space missions are needed to develop. Extreme environments are good scenarios where to test and train those techniques and where hypothetical astrobiological space missions could be simulated for increasing possibilities of micro niches identification.

AbstractRecently reported results from latest Mars Orbiters and Rovers missions are transforming our opinion about the red planet. That dry and inhospitable planet reported in the past is becoming a wetter planet with high probabilities of water existence in the past. Nowadays, some results seem to indicate the presence of water beneath the Mars surface. But also mineralogy studies by NASA Opportunity Rover report iron oxides and hydroxides precipitates on Endurance Crater. Sedimentary deposits have been identified at Meridiani Planum. These deposits must have generated in a dune aqueous acidic and oxidizing environment. Similarities appear when we study Rio Tinto, and acidic river under the control of iron.The discovery of extremophiles on Earth widened the window of possibilities for life to develop in the Universe, and as a consequence on Mars and other planetary bodies with astrobiological interest. The compilation of data produced by the ongoing missions offers an interested view for life possibilities to exist: signs of an early wet Mars and rather recent volcanic activity as well as ground morphological characteristics that seem to be promoted by liquid water. The discovery of important accumulations of sulfates and the existence of iron minerals such as jarosite in rocks of sedimentary origin has allowed specific terrestrial models to come into focus. Río Tinto (Southwestern Spain, Iberian Pyritic Belt) is an extreme acidic environment, product of the chemolithotrophic activity of micro-organisms that thrive in the massive pyrite-rich deposits of the Iberian Pyritic Belt. Some particular protective environments should house the organic molecules and bacterial life forms in harsh environments such as Mars surface supporting microniches inside precipitated minerals or inside rocks. Terrestrial analogues could help us to afford the comprehension of habitability (on other planetary bodies).We are reporting here the multidisciplinary study of some endolithic niches inside salt deposits used by phototrophs for taking advantage of sheltering particular light wavelengths. These acidic salts deposits located in Río Tinto shelter life forms that are difficult to visualize by eye. This interdisciplinary field analogue campaign was conducted in the framework of the CAREX FP7 EC programme.

The recent geomicrobiological characterization of Río Tinto, Iberian Pyrite Belt (IPB), has proven the importance of the iron cycle, not only in generating the extreme conditions of the habitat (low pH, high concentration of toxic heavy metals) but also in maintaining the high level of microbial diversity, both prokaryotic and eukaryotic, detected in the water column and the sediments. The extreme conditions of the Tinto basin are not the product of industrial contamination but the consequence of the presence of an underground bioreactor that obtains its energy from the massive sulfide minerals of the IPB. To test this hypothesis, a drilling project was carried out to intersect ground waters that interact with the mineral ore in order to provide evidence of subsurface microbial activities and the potential resources to support these activities. The oxidants that drive the system appear to come from the rock matrix, contradicting conventional acid mine drainage models. These resources need only groundwater to launch microbial metabolism. There are several similarities between the vast deposits of sulfates and iron oxides on Mars and the main sulfide-containing iron bioleaching products found in the Tinto. Firstly, the short-lived methane detected both in Mars' atmosphere and in the sediments and subsurface of the IPB and secondly, the abundance of iron, common to both. The physicochemical properties of iron make it a source of energy, a shield against radiation and oxidative stress as well as a natural pH controller. These similarities have led to Río Tinto's status as a Mars terrestrial analogue.