Saline lakes are expected to have been extensively present on ancient Mars, particularly as the planet dried or cooled. Such lakes likely deposited sulfate salts, as these salts have been widely identified from orbital and in situ Mars data. However, the relationship between martian sulfates and the environmental conditions that formed them (including whether conditions were warm, cool, drying, or freezing), remains under-characterized. To evaluate the relationship between sulfates and climate, we investigated the hypersaline, sulfate-bearing Basque Lakes in British Columbia, Canada, which serve as an analog for both "cold and wet" and "warm and wet" early Mars. We use the rover and orbiter relevant instrument techniques of Raman, Near Infrared, and X-Ray Fluorescence spectroscopy to evaluate seasonal lake mineralogy. We find that temperature-dependent, multi-cation salts form widely within the Basque Lakes' efflorescent crusts in the fall, which transform to meridianiite, mirabilite, a metastable Na-sulfate 7-hydrate, and epsomite in the wintertime. In both seasons, salt assemblages are metastable and persist beyond expected thermodynamic stability fields, suggesting ongoing climatic changes can prevent saline systems from settling into equilibrium phases. Coupled with sedimentological evidence, intimately mixed Mg-sulfates of different hydration states could be an indicator of surface fluids that interacted with an atmosphere, while formation of Na-sulfates could be evidence for brine freezing. Curiosity's exploration of the Gale crater sulfate-bearing unit and Perseverance's exploration of Jezero crater on Mars offer excellent chances to investigate the influence of climate on Mg-sulfate formation.

Concentrated magnesium chloride brines are extreme environments that are inhospitable to life on Earth. The ionic strength of these brines significantly depresses water activity and concomitantly exerts significant chaotropic stress. Although these brines are largely considered sterile, the well-known preservative effects of magnesium chloride on certain biomolecules, such as DNA, confound life detection approaches and efforts to constrain precisely the habitable window of life on Earth. While the ability of these brines to preserve genetic material is well documented, the preservation of whole cells, which are generally thought to be preserved in magnesium chloride brines, is poorly described. This work explores the effects of long-term exposure of highly chaotropic magnesium chloride on viability, cell integrity, and DNA preservation in the model organisms Escherichia coli, Salinibacter ruber, Halobacterium salinarum, and Haloquadratum walsbyi. The selected halophiles are relevant for this study as they are abundant and globally distributed in brine environments, while E. coli was chosen to represent infall or transport of non-adapted cells. We observed unexpected resilience in E. coli, which survived in 4 M magnesium chloride for longer than the tested halophiles, and nonviable cells maintained structural whole-cell integrity for over 3 years. Whole S. ruber cells were also preserved in 4 M magnesium chloride, while the tested haloarchaea lost viability and completely degraded within hours of exposure. DNA from all tested strains was recovered from incubations after upwards of 3 years of exposure; it showed some signs of degradation but was nonetheless still amplifiable via polymerase chain reaction. Our work demonstrates that the preservation of whole cells in magnesium chloride brines is not universal. Considering the potential abundance of chaotropic brine environments within our solar system, understanding the limits of life and the preservation of biosignatures in these brines is critical to inform future life detection missions on Earth and beyond.

The transition from day to night brings sweeping change to both environments and the organisms within them. Diel shifts in gene expression have been documented across all domains of life but remain understudied in microbial communities, particularly those in extreme environments where small changes may have rippling effects on resource availability. In hypersaline environments, many prominent taxa are photoheterotrophs that rely on organic carbon for growth but can also generate significant ATP via light-powered rhodopsins. Previous research demonstrated a significant response to light intensity shifts in the model halophile Halobacterium salinarum, but these cycles have rarely been explored in situ. Here, we examined genome-resolved differential expression in a hypersaline saltern (water activity (aw) $$\cong$$ 0.83, total dissolved solids = 250.7 g L−1) throughout a 24-h period. We found increased transcription of genes related to phototrophy and anabolic metabolic processes during the day, while genes related to aerobic respiration and oxidative stress were upregulated at night. Substantiating these results with a chemostat culture of the environmentally abundant halophilic bacterium Salinibacter ruber revealed similar transcriptional upregulation of genes associated with aerobic respiration under dark conditions. These results describe the potential for light-driven changes in oxygen use across both a natural hypersaline environment and a pure culture. Whole-community survey of diel changes in gene expression in a hypersaline saltern indicates metabolic shifts that influence patterns of oxygen use, highlighting the intertwined role of microbial and geochemical processes in shaping the environment.

Modern advancements in laboratory and instrumental techniques in astrobiology have improved our life detection capabilities on both Earth and beyond. These advancements have also increased the complexity of data often resulting in datasets that are characterized by complex and non-linear relationships. Machine learning methods are underutilized in astrobiology; however, these methods are extremely effective at revealing structure and patterns in complex datasets when paired with the right algorithms. Here, we employ a series of classification and regression algorithms to predict the abundance of organic carbon (OC) from X-ray fluorescence (XRF) data in dynamic Mars-analog hypersaline lake sediments. More specifically, we constructed models using the random forest (RF), k-nearest neighbors (KNN), support vector machine (SVM), and logistic regression (LR) algorithms. Overall, our trained models showed good performance with predicting the abundance of OC, with accuracies from 80% to 94%. Our results show how applying predictive models to astrobiology datasets can help life detection efforts. Machine learning approaches such as classification and regression algorithms offer insight into complex data while providing agnostic insights, ultimately creating a more efficient search for OC. We applied our trained model on XRF data from Martian soil using PIXL and Odyssey datasets to produce probability predictions of OC abundance. Our predictions show a high probability that OC abundance is low which is comparable to OC data from recently landed missions. These results highlight the potential for machine learning models to be trained on data from analog environments on Earth and then transferred (transfer learning) to extraterrestrial targets.

Impacts are critical to producing the aqueous environments necessary to stimulate prebiotic chemistry on Titan’s surface. Furthermore, organic hazes resting on the surface are a likely feedstock of biomolecules. In this work, we conduct impact experiments on laboratory-produced organic haze particles and haze/sand mixtures and analyze these samples for life’s building blocks. Samples of unshocked haze and sand particles are also analyzed to determine the change in biomolecule concentrations and distributions from shocking. Across all samples, we detect seven nucleobases, nine proteinogenic amino acids, and five other biomolecules (e.g., urea) using a blank subtraction procedure to eliminate signals due to contamination. We find that shock pressures of 13 GPa variably degrade nucleobases, amino acids, and a few other organics in haze particles and haze/sand mixtures; however, certain individual biomolecules become enriched or are even produced from these events. Xanthine, threonine, and aspartic acid are enriched or produced in impact experiments containing sand, suggesting these minerals may catalyze the production of these biomolecules. On the other hand, thymine and isoleucine/norleucine are enriched or produced in haze samples containing no sand, suggesting catalytic grains are not necessary for all impact shock syntheses. Uracil, glycine, proline, cysteine, and tyrosine are the most unstable to impact-related processing. These experiments suggest that impacts alter biomolecule distributions on Titan’s surface, and that organic hazes co-occurring with fine-grained material on the surface may provide an initial source for further prebiotic chemistry on Titan.

Evidence for the beneficial role of impacts in the creation of urable or habitable environments on Earth prompts the question of whether meteorite impacts could play a similar role at other potentially urable/habitable worlds like Enceladus, Europa, and Titan. In this work, we demonstrate that to first order, impact conditions on these worlds are likely to have been consistent with the survival of organic compounds and/or sufficient for promoting synthesis in impact melt. We also calculate melt production and freezing times for crater sizes found at Enceladus, Europa, and Titan and find that even the smallest craters at these worlds offer the potential to study the evolution of chemical pathways within impact melt. These first-order calculations point to a critical need to investigate these processes at higher fidelity with lab experiments, sophisticated thermodynamic and chemical modeling, and, eventually, in situ investigations by missions.

The abundance of potentially habitable hypersaline environments in our solar system compels us to understand the impacts of high-salt matrices and brine dynamics on biosignature detection efforts. We identified and quantified organic compounds in brines from South Bay Salt Works (SBSW), where evapoconcentration of ocean water enables exploration of the impact of NaCl- and MgCl2-dominated brines on the detection of potential biosignature molecules. In SBSW, organic biosignature abundance and distribution are likely influenced by evapoconcentration, osmolyte accumulation, and preservation effects. Bioluminescence assays show that adenosine triphosphate (ATP) concentrations are higher in NaCl-rich, low water activity (aw) samples (<0.85) from SBSW. This is consistent with the accumulation and preservation of ATP at low aw as described in past laboratory studies. The water-soluble small organic molecule inventory was determined by using microchip capillary electrophoresis paired with high-resolution mass spectrometry (µCE-HRMS). We analyzed the relative distribution of proteinogenic amino acids with a recently developed quantitative method using CE-separation and laser-induced fluorescence (LIF) detection of amino acids in hypersaline brines. Salinity trends for dissolved free amino acids were consistent with amino acid residue abundance determined from the proteome of the microbial community predicted from metagenomic data. This highlights a tangible connection up and down the "-omics" ladder across changing geochemical conditions. The detection of water-soluble organic compounds, specifically proteinogenic amino acids at high abundance (>7 mM) in concentrated brines, demonstrates that potential organic biomarkers accumulate at hypersaline sites and suggests the possibility of long-term preservation. The detection of such molecules in high abundance when using diverse analytical tools appropriate for spacecraft suggests that life detection within hypersaline environments, such as evaporates on Mars and the surface or subsurface brines of ocean world Europa, is plausible and argues such environments should be a high priority for future exploration. Key Words: Salts-Analytical chemistry-Amino acids-Biosignatures-Capillary electrophoresis-Preservation. Astrobiology 24, 795-812.

The environmental conditions on present-day Mars are detrimental for life as we know it; however cumulative evidence suggests that early Noachian Mars (~ 4 billion Ga) had a warmer climate with a denser atmosphere1 capable of supporting surficial liquid water, and providing protection from UV and cosmic radiation. It is possible, therefore, that early Mars could have been hospitable for microorganisms.While cellular degeneration is a rapid process following cell death, microorganisms do leave behind molecular clues as to their existence, or biosignatures, such as the lipid molecules that previously comprised their cell membranes. Each group of microorganisms leaves behind a distinct lipid “fingerprint” that is relatively resistant to harsh environmental conditions and can be preserved over geological timescales2.Various robotic and remote-sensing missions have confirmed the presence of salt deposits (e.g., chlorides, sulfates) at the late Noachian to late Hesperian (~ 4 to 3.5 Ga) boundaries of present-day Martian deposits. In particular, bulk enrichments of calcium and magnesium sulfates have been reported in the Hesperian (~ 3.3–3.7 Ga) sedimentary rocks of Gale crater3, which indicate the previous presence of widespread liquid water on Mars. On Earth, such salts have been found to harbour and protect microbial life for a prolonged period, possibly over millions of years4, thus evaporite sequences provide a compelling target for life detection, as putative biosignatures could be preserved5.Currently, the beneficial or deleterious effects of sulfate chemistry on the preservation of organic matter, especially under biologically destructive modern Martian conditions is not well constrained. Given the prominent presence of sulfates on the surface of Mars, this work explores whether sulfate minerals can act as viable substrates for the long-term preservation of lipids, when exposed to Mars-like radiative and atmospheric conditions, and aims to determine whether those signs of life can be explicitly detected.To carry out this work, we use a combination of analogue fieldwork and laboratory-based simulation studies. Microorganisms isolated from a magnesium sulfate-rich analogue site (Basque Lake) and other terrestrial early Mars analogues, will be entombed within artificial sulfate crystals by evaporating an experimental brine under low atmospheric pressure and UV radiation similar to that encountered on the surface of Mars using The Open University’s Mars chamber facility. The lipids within the samples will be extracted and derivatized to make them amenable to by pyrolysis-gas chromatography-mass spectrometry (py-GC-MS); py-GC-MS is an important component of the Sample Analysis at Mars (SAM) instrument suite onboard NASA’s Mars Science Laboratory (MSL) and of the Mars Organic Molecular Organizer (MOMA) on the ESA’s Rosalind Franklin (ExoMars) rover. This work will aid in characterizing the impacts of the simulated Martian environment and Martian mineralogy on the preservation and detection of lipid biomarkers within sulfitic evaporite deposits and will inform the robotic missions targeting the search for ancient signatures of life on Mars.References: [1] Carr MH, Head JW (2010) Earth and Planetary Science Letters 294: 185-203 [2] Luo G et al., (2019) Earth-Science Reviews 189: 99-124 [3] Rapin, W et al., (2019) Nature Geosciences 12:889–895 [4] Fendrihan S et al., (2006) Reviews in Environmental Science and Biotechnology 5: 203-218 [5] Johnson SS et al., (2020) Astrobiology 2020 20:167-178

Hypersaline brines provide excellent opportunities to study extreme microbial life. Here, we investigated anabolic activity in nearly 6000 individual cells from solar saltern sites with water activities ( a w ) ranging from 0.982 to 0.409 (seawater to extreme brine). Average anabolic activity decreased exponentially with a w , with nuanced trends evident at the single-cell level: The proportion of active cells remained high (>50%) even after NaCl saturation, and subsets of cells spiked in activity as a w decreased. Intracommunity heterogeneity in activity increased as seawater transitioned to brine, suggesting increased phenotypic heterogeneity with increased physiological stress. No microbial activity was detected in the 0.409- a w brine (an MgCl 2 -dominated site) despite the presence of cell-like structures. Extrapolating our data, we predict an a w limit for detectable anabolic activity of 0.540, which is beyond the currently accepted limit of life based on cell division. This work demonstrates the utility of single-cell, metabolism-based techniques for detecting active life and expands the potential habitable space on Earth and beyond.

Modern and ancient hypersaline lakes and oceans have been identified across the solar system, but the habitability and potential of these environments to preserve organic matter remain unknown. Here, we evaluate organic matter production and preservation potential in hypersaline lakes whose chemistries resemble deposits on Mars. We focus our analysis on lipid biomarkers including fatty acids, alkanes, and ether-bound lipids in modern brines, salt deposits, and surface sediments. We also report total organic carbon (TOC), carbon/nitrogen (C/N) ratios, and bulk OC (δ13C and δ15N) isotopes to contextualize the lipid data. In all lakes, the predominant biosignatures include short chain fatty acids (C<23) suggesting microbial origin. Sediments also incorporate a diversity of microbially and terrestrially derived lipids. Ether-bound lipids derived from archaea and bacteria constitute a minor but measurable fraction of the lipids in brines. This result contrasts with typical results from NaCl brines which contain significant archaeal biomass. TOC concentrations in sediments are universally high, ranging from 0.7% to 12% with sulfate-rich sediments having the highest concentrations. The isotopic composition of TOC corroborates the biomarker results, showing δ13C values and C/N values indicative of aquatic microbial origin. This richness of organic material and in situ microbial biosignatures differ from previously studied Cl-dominated Mars-analog sites which have shown limited organic matter production and preservation and acidic SO4-rich hypersaline environments which were dominated by terrestrial inputs. Overall, our results suggest that Mg-SO4-rich hypersaline environments harbor a rich microbial biomarker landscape and are ideal locations for preserving these signatures, potentially over geological timescales.

Modern and ancient hypersaline brines have been identified across the solar system, but the habitability of these environments remains unknown. Here, we evaluate organic matter (OM) production in MgSO 4 and Na 2 CO 3 rich hypersaline lakes whose chemistries resemble deposits on Mars such as those identified in Jezero crater. We focus our analysis on lipid biomarkers including fatty acids, alkanes, and ether‐bound lipids in modern brines, salt deposits, and surface sediments. We also report total organic carbon (TOC), carbon/nitrogen (C/N) ratios, and bulk OC (δ 13 C and δ 15 N) isotopes to contextualize the lipid biomarker data. In all lakes, the predominant biosignatures include midchain (12 < C < 23) fatty acids and alkanes suggesting microbial origin. Sediments also incorporate a greater diversity of lipids. Ether‐bound lipids derived from archaea and bacteria constitute a minor but measurable fraction of the lipids. This result contrasts with typical findings from other studies with NaCl brines which contain significant archeal biomass. TOC concentrations in all sediments are high, ranging from 0.7% to 12% with sulfate‐rich sediments having the highest concentrations. The isotopic and elemental compositions of TOC corroborate the biomarker results, showing δ 13 C values and C/N values indicative of aquatic microbial origin. This richness of organic matter and in situ microbial biosignatures differ from previously studied Cl‐dominated Mars‐analog sites which have shown limited OM production and preservation and acidic SO 4 ‐rich hypersaline environments which were dominated by terrestrial inputs. Overall, our results suggest that MgSO 4 ‐rich hypersaline environments are conducive for life and harbor a rich microbial biomarker landscape.

We present thermophysical, biological, and chemical observations of ice and brine samples from five compositionally diverse hypersaline lakes in British Columbia's interior plateau. Possessing a spectrum of magnesium, sodium, sulfate, carbonate, and chloride salts, these low-temperature high-salinity lakes are analogs for planetary ice-brine environments, including the ice shells of Europa and Enceladus and ice-brine systems on Mars. As such, understanding the thermodynamics and biogeochemistry of these systems can provide insights into the evolution, habitability, and detectability of high-priority astrobiology targets. We show that biomass is typically concentrated in a layer near the base of the ice cover, but that chemical and biological impurities are present throughout the ice. Coupling bioburden, ionic concentration, and seasonal temperature measurements, we demonstrate that impurity entrainment in the ice is directly correlated to ice formation rate and parent fluid composition. We highlight unique phenomena, including brine supercooling, salt hydrate precipitation, and internal brine layers in the ice cover, important processes to be considered for planetary ice-brine environments. These systems can be leveraged to constrain the distribution, longevity, and habitability of low-temperature solar system brines-relevant to interpreting spacecraft data and planning future missions in the lens of both planetary exploration and planetary protection.

Concurrent osmotic and chaotropic stress make MgCl 2 -rich brines extremely inhospitable environments. Understanding the limits of life in these brines is essential to the search for extraterrestrial life on contemporary and relict ocean worlds, like Mars, which could host similar environments. We sequenced environmental 16S rRNA genes and quantified microbial activity across a broad range of salinity and chaotropicity at a Mars-analogue salt harvesting facility in Southern California, where seawater is evaporated in a series of ponds ranging from kosmotropic NaCl brines to highly chaotropic MgCl 2 brines. Within NaCl brines, we observed a proliferation of specialized halophilic Euryarchaeota, which corresponded closely with the dominant taxa found in salterns around the world. These communities were characterized by very slow growth rates and high biomass accumulation. As salinity and chaotropicity increased, we found that the MgCl 2 -rich brines eventually exceeded the limits of microbial activity. We found evidence that exogenous genetic material is preserved in these chaotropic brines, producing an unexpected increase in diversity in the presumably sterile MgCl 2 -saturated brines. Because of their high potential for biomarker preservation, chaotropic brines could therefore serve as repositories of genetic biomarkers from nearby environments (both on Earth and beyond) making them prime targets for future life-detection missions.

Introduction: The environmental conditions on present-day Mars (mean surface temperature −60°C, atmospheric pressure less than 1% of Earth’s, and high levels of solar UV and ionizing radiation) are detrimental for life as we know it; however cumulative evidence suggests that early Noachian Mars (~ 4 Gyr) had a warmer climate with a denser atmosphere [1], capable of supporting surficial liquid water [2] and providing protection from UV and ionizing radiation. The widespread aqueous systems on the surface of early Mars (e.g., saline lakes, evaporative ponds) appear astrobiologically promising [3] and their presence coincides with the time when life emerged on Earth [4]. Therefore, the habitability of martian paleoenvironments is the prime motivation for the search for life on Mars [5]. The presence of evaporite deposits (e.g., chlorides, sulfates, and perchlorates) has been confirmed on the ancient surfaces of Mars (Noachian to Hesperian, 4 to 3.7 billion years old [6,7]). A recent study reported high concentrations of hydrated Mg-sulfate (26–36 wt%) and Ca-sulfate (30–50 wt%) within the Gale crater lacustrine deposits, as identified by the ChemCam laser-induced breakdown spectrometer and other instruments onboard the Curiosity rover [6]. These deposits are thought to have precipitated from the brines, possibly at the beginning of the Hesperian era (~3.7 Gyr), when they evaporated. It is possible that remnants of microbial life present on early Mars could have been preserved within these salts; on Earth, fluid inclusions within crystalline salts have been found to harbour and protect microbial life for a prolonged period, possibly over millions of years [8]. Thus, evaporite sequences provide a compelling target for life detection in which putative biosignatures could be preserved [9]. The residual organic biosignatures (e.g., longchain hydrocarbons) produced via metabolism and reproduction in living systems [10] can be preserved and detected even after significant geological time has passed since it deceased [11]. Among such biosignatures, the fatty acid components of the lipid molecules that constitute cell membranes are deemed less prone to environmental deterioration and are compatible with long-term preservation [12]. Given the prominence of sulfates on the surface of Mars, this work explores whether sulfate minerals can act as viable substrates for the long-term preservation of fatty acids when exposed to Mars-like radiative and atmospheric conditions. This is particularly important because the beneficial or deleterious effects of sulfate chemistry on the preservation of organic matter, especially under biologically destructive modern martian conditions, is not well constrained. The work also aims to determine whether these biosignatures can be explicitly detected by spectroscopic instruments. Methods: We have isolated microorganisms from the evaporite assemblages of Basque Lakes (BL) (Figure 1), south-central British Columbia, Canada. These lakes are saturated with MgSO4 and considered analogous to early Mars lacustrine environments. The seasonal mean temperature of the valley oscillates from 10°C (January) to 25°C/30°C (July/August) [13].

Deep-sea hypersaline anoxic basins (DHABs) are uniquely stratified polyextreme environments generally found in enclosed seas. These environments select for elusive and widely uncharacterized microbes that may be living below the currently recognized window of life on Earth. Still, there is strong evidence of highly specialized active microbial communities in the Kryos, Discovery, and Hephaestus basins located in the Eastern Mediterranean Sea; the only known athalassohaline DHABs. Life is further constrained in these DHABs as near-saturated concentrations of magnesium chloride significantly reduces water activity (aw ) and exerts extreme chaotropic stress, the tendency of a solution to disorder biomolecules. In this review, we provide an overview of microbial adaptations to polyextremes focusing primarily on chaotropicity, summarize current evidence of microbial life within athalassohaline DHABs and describe the difficulties of life detection approaches and sampling within these environments. We also reveal inconsistent measurements of chaotropic activity in the literature highlighting the need for a new methodology. Finally, we generate recommendations for future investigations and discuss the importance of athalassohaline DHAB research to help inform extraterrestrial life detection missions.

Emma K. Brown, Jacob J. Buffo, Meg Grantham, Alexandra Pontefract, Jennifer Glass, Ellery Ingall, Peter Doran, Mario Toubes-Rodrigo, Hannah Dion-Kirschner, Chris Carr, Jeff S. Bowman, Britney E. Schmidt, and the OAST Team. Georgia Institute of Technology (emma.brown@gatech.edu) Darmouth College, Georgetown University, LSU, Open University, Northwestern University, Massachusetts Institute of Technology, Scripps Institute of Oceanography