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Identifying unequivocal signs of life on Mars is one of the most important objectives for sending missions to the red planet. Here we report Red Stone, a 163-100 My alluvial fan-fan delta that formed under arid conditions in the Atacama Desert, rich in hematite and mudstones containing clays such as vermiculite and smectites, and therefore geologically analogous to Mars. We show that Red Stone samples display an important number of microorganisms with an unusual high rate of phylogenetic indeterminacy, what we refer to as "dark microbiome", and a mix of biosignatures from extant and ancient microorganisms that can be barely detected with state-of-the-art laboratory equipment. Our analyses by testbed instruments that are on or will be sent to Mars unveil that although the mineralogy of Red Stone matches that detected by ground-based instruments on the red planet, similarly low levels of organics will be hard, if not impossible to detect in Martian rocks depending on the instrument and technique used. Our results stress the importance in returning samples to Earth for conclusively addressing whether life ever existed on Mars., (© 2023. The Author(s).)

We present the hypothesis that microorganisms can change the freezing/melting curve of cold salty solutions by protein expression, as it is known that proteins can affect the liquid-to-ice transition, an ability that could be of ecological advantage for organisms on Earth and on Mars. We tested our hypothesis by identifying a suitable candidate, the well-known psycrophile and halotolerant bacteria Rhodococcus sp. JG3, and analyzing its response in culture conditions that included specific hygroscopic salts relevant to Mars-that is, highly concentrated magnesium perchlorate solutions of 20 wt % and 50 wt % Mg(ClO 4 ) 2 at both end members of the eutectic concentration (44 wt %)-and subfreezing temperatures (263 K and 253 K). Using a combination of techniques of molecular microbiology and aqueous geochemistry, we evaluated the potential roles of proteins over- or underexpressed as important players in different mechanisms for the adaptability of life to cold environments. We recorded the changes observed by micro-differential scanning calorimetry. Unfortunately, Rhodococcus sp. JG3 did not show our hypothesized effect on the melting characteristics of cold Mg-perchlorate solutions. However, the question remains as to whether our novel hypothesis that halophilic/psychrophilic bacteria or archaea can alter the freezing/melting curve of salt solutions could be validated. The null result obtained after analyzing just one case lays the foundation to continue the search for proteins produced by microorganisms that thrive in very cold, high-saline solutions, which would involve testing different microorganisms with different salt components. The immediate implications for the habitability of Mars are discussed.

The aim of the European Space Agency's ExoMars rover mission is to search for potential traces of present or past life in the swallow subsurface (2 m depth) of Mars. The ExoMars rover mission relies on a suite of analytical instruments envisioned to identify organic compounds with biological value (biomarkers) associated with a mineralogical matrix in a highly oxidative environment. We investigated the feasibility of detecting basic organics (linear and branched lipid molecules) with Raman laser spectroscopy, an instrument onboard the ExoMars rover, when exposed to oxidant conditions. We compared the detectability of six lipid molecules (alkanes, alkanols, fatty acid, and isoprenoid) before and after an oxidation treatment (15 days with hydrogen peroxide), with and without mineral matrix support (amorphous silica rich vs. iron rich). Raman and infrared spectrometry was combined with gas chromatography-mass spectrometry to determine detection limits and technical constraints. We observed different spectral responses to degradation depending on the lipid molecule and mineral substrate, with the silica-rich material showing better preservation of organic signals. These findings will contribute to the interpretation of Raman laser spectroscopy results on cores from the ExoMars rover landing site, the hydrated silica-enriched delta fan on Cogoon Vallis (Oxia Planum).

The search for biomarkers of present or past life is one of the major challenges for in situ planetary exploration. Multiple constraints limit the performance and sensitivity of remote in situ instrumentation. In addition, the structure, chemical, and mineralogical composition of the sample may complicate the analysis and interpretation of the results. The aim of this work is to highlight the main constraints, performance, and complementarity of several techniques that have already been implemented or are planned to be implemented on Mars for detection of organic and molecular biomarkers on a best-case sample scenario. We analyzed a 1000-year-old desiccated and mummified microbial mat from Antarctica by Raman and IR (infrared) spectroscopies (near- and mid-IR), thermogravimetry (TG), differential thermal analysis, mass spectrometry (MS), and immunological detection with a life detector chip. In spite of the high organic content (ca. 20% wt/wt) of the sample, the Raman spectra only showed the characteristic spectral peaks of the remaining beta-carotene biomarker and faint peaks of phyllosilicates over a strong fluorescence background. IR spectra complemented the mineralogical information from Raman spectra and showed the main molecular vibrations of the humic acid functional groups. The TG-MS system showed the release of several volatile compounds attributed to biopolymers. An antibody microarray for detecting cyanobacteria (CYANOCHIP) detected biomarkers from Chroococcales, Nostocales, and Oscillatoriales orders. The results highlight limitations of each technique and suggest the necessity of complementary approaches in the search for biomarkers because some analytical techniques might be impaired by sample composition, presentation, or processing. Key Words: Planetary exploration-Life detection-Microbial mat-Life detector chip-Thermogravimetry-Raman spectroscopy-NIR-DRIFTS. Astrobiology 17, 984-996.

The particular mineralogy formed in the acidic conditions of the Río Tinto has proven to be a first-order analogue for the acid-sulfate aqueous environments of Mars. Therefore, studies about the formation and preservation of biosignatures in the Río Tinto will provide insights into equivalent processes on Mars. We characterized the biomolecular patterns recorded in samples of modern and old fluvial sediments along a segment of the river by means of an antibody microarray containing more than 200 antibodies (LDCHIP200, for Life Detector Chip) against whole microorganisms, universal biomolecules, or environmental extracts. Samples containing 0.3-0.5 g of solid material were automatically analyzed in situ by the Signs Of LIfe Detector instrument (SOLID2), and the results were corroborated by extensive analysis in the laboratory. Positive antigen-antibody reactions indicated the presence of microbial strains or high-molecular-weight biopolymers that originated from them. The LDCHIP200 results were quantified and subjected to a multivariate analysis for immunoprofiling. We associated similar immunopatterns, and biomolecular markers, to samples with similar sedimentary age. Phyllosilicate-rich samples from modern fluvial sediments gave strong positive reactions with antibodies against bacteria of the genus Acidithiobacillus and against biochemical extracts from Río Tinto sediments and biofilms. These samples contained high amounts of sugars (mostly polysaccharides) with monosaccharides like glucose, rhamnose, fucose, and so on. By contrast, the older deposits, which are a mix of clastic sands and evaporites, showed only a few positives with LDCHIP200, consistent with lower protein and sugar content. We conclude that LDCHIP200 results can establish a correlation between microenvironments, diagenetic stages, and age with the biomarker profile associated with a sample. Our results would help in the search for putative martian biomarkers in acidic deposits with similar diagenetic maturity. Our LDCHIP200 and SOLID-like instruments may be excellent tools for the search for molecular biomarkers on Mars or other planets.

A search for evidence of cryptic life in the subsurface region of a fractured Paleozoic volcanosedimentary deposit near the source waters of the Río Tinto River (Iberian pyrite belt, southwest Spain) was carried out by Mars Astrobiology Research and Technology Experiment (MARTE) project investigators in 2003 and 2004. This conventional deep-drilling experiment is referred to as the MARTE ground truth drilling project. Boreholes were drilled at three sites, and samples from extracted cores were analyzed with light microscopy, scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy. Core leachates were analyzed with ion chromatography, and borehole fluids were analyzed with ion and gas chromatography. Key variables of the groundwater system (e.g., pO(2), pH, and salinity) exhibit huge ranges probably due to surficial oxygenation of overall reducing waters, physical mixing of waters, and biologically mediated water-rock interactions. Mineral distribution is mainly driven by the pH of subsurface solutions, which range from highly acidic to neutral. Borehole fluids contain dissolved gases such as CO(2), CH(4), and H(2). SEM-EDS analyses of core samples revealed evidence of microbes attacking pyrite. The Río Tinto alteration mechanisms may be similar to subsurface weathering of the martian crust and provide insights into the possible (bio)geochemical cycles that may have accompanied underground habitats in extensive early Mars volcanic regions and associated sulfide ores.

Recent data from space missions reveal that there are ongoing climatic changes and erosive processes that continuously modify surface features of Mars. We have investigated the seasonal dynamics of a number of morphological features located at Inca City, a representative area at high southern latitude that has undergone seasonal processes. By integrating visual information from the Mars Orbiter Camera on board the Mars Global Surveyor and climatic cycles from a Mars' General Circulation Model, and considering the recently reported evidence for the presence of water-ice and aqueous precipitates on Mars, we propose that a number of the erosive features identified in Inca City, among them spiders, result from the seasonal melting of aqueous salty solutions.