Current state of athalassohaline deep-sea hypersaline anoxic basin research-recommendations for future work and relevance to astrobiology.

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.

Microbial diversity and activity in Southern California salterns and bitterns: analogues for remnant ocean worlds.

Concurrent osmotic and chaotropic stress make MgCl2 -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 MgCl2 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 MgCl2 -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 MgCl2 -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.

Microbial Life in Impact Craters.

Asteroid and comet impacts are known to have caused profound disruption to multicellular life, yet their influence on habitats for microorganisms, which comprise the majority of Earth's biomass, is less well understood. Of particular interest are geological changes in the target lithology at and near the point of impact that can persist for billions of years. Deep subsurface and surface-dwelling microorganisms are shown to gain advantages from impact-induced fracturing of rocks. Deleterious changes are associated with impact-induced closure of pore spaces in rocks. Superimposed on these long-term geological changes are post-impact alterations such as changes in the hydrological system in and around a crater. The close coupling between geological changes and the conditions for microorganisms yields a synthesis of the fields of microbiology and impact cratering. We use these data to discuss how craters can be used in the search for life beyond Earth.

Biosignature Molecules Accumulate and Persist in Evaporitic Brines: Implications for Planetary Exploration.

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.

Single-cell analysis in hypersaline brines predicts a water-activity limit of microbial anabolic activity.

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 (aw) ranging from 0.982 to 0.409 (seawater to extreme brine). Average anabolic activity decreased exponentially with aw, 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 aw 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-aw brine (an MgCl2-dominated site) despite the presence of cell-like structures. Extrapolating our data, we predict an aw 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.

The Bioburden and Ionic Composition of Hypersaline Lake Ices: Novel Habitats on Earth and Their Astrobiological Implications.

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.

Sequencing nothing: Exploring failure modes of nanopore sensing and implications for life detection.

The detection of extant life is a major focus of many planned future planetary missions, a current challenge of which is the ability to target biomarkers capable of providing unambiguous evidence of life. DNA sequencing is increasingly recognized as a powerful tool for life detection for planetary exploration missions; beyond use of sequence information to determine the origins of the sample (e.g., extant life or forward contamination), recent advances in the field have enabled interrogation of single molecules, with or without amplification. The focus of this work is on failure modes, specifically the issues encountered when there is no-to-low input DNA into a sequencing device, and the potential for the generation of sequencing artifacts that could be interpreted as a false positive. Using Oxford Nanopore Technologies (ONT) MinION, we assess whether single molecule sequencing, involving no amplification, generates noise signals that could be misinterpreted in the context of a planetary exploration mission, and also whether the ability of the instrument to handle these types of situations could make it feasible for clean room monitoring. Utilizing quality score filtering techniques in place at the time of this experiment, runs containing only initial flowcell chemistry and/or library reagents generated 5 passing reads out of a total of 3568 measured reads, and contained estimated sequences with low complexity that did not map to the NCBI database. The noise characteristics in all instances suggest that quality thresholds were appropriately chosen by ONT: new chemistry and basecalling workflows have shown further suppression of noise sources, which completely mitigate the generation of spurious reads.

Characterization of Microbial Communities Hosted in Quartzofeldspathic and Serpentinite Lithologies in Jeffrey Mine, Canada.

The microbial ecology and activity of serpentine deposits and associated hydrated minerals are largely unknown. Previous research has largely focused on microbial communities in active serpentinizing systems, whereas relatively little research has demonstrated the ability of serpentine deposits to host microbial communities after the cessation of serpentinization. Given the potential role of serpentinization reactions fueling primitive microbial metabolisms on early Earth and the identification of serpentine deposits on Mars, knowledge of these geobiological relationships and potential for serpentine to host extant microbial communities and preserve biosignatures is increasingly important for planetary exploration seeking signs of life. The selection of habitable sites most likely to yield putative biosignatures is crucial to mission success. In this study, we aimed to characterize, on the basis of both metabolic activity and taxonomic composition, the microbial communities hosted in two naturally co-occurring and mineralogically distinct substrates within the serpentine-rich Jeffrey Mine pit-igneous quartzofeldspathic intrusives and serpentinite. Detection of heterotrophic activity in both lithologies at 24°C, and in serpentinite at -5°C, demonstrated that each substrate had the ability to host a viable microbial community, at Mars-relevant temperatures. Targeted amplicon sequencing subsequently showed the presence of bacterial, fungal, and photosynthetic microbial communities in both substrates. Here, we have demonstrated the presence of a viable lithic microbial community within two rock types in the Jeffrey Mine and provided evidence that lithologies associated with serpentine deposits and proximal hydrated minerals have the ability to support diverse prokaryotic and eukaryotic microbial colonization.

Microbial Diversity in a Hypersaline Sulfate Lake: A Terrestrial Analog of Ancient Mars.

Life can persist under severe osmotic stress and low water activity in hypersaline environments. On Mars, evidence for the past presence of saline bodies of water is prevalent and resulted in the widespread deposition of sulfate and chloride salts. Here we investigate Spotted Lake (British Columbia, Canada), a hypersaline lake with extreme (>3 M) levels of sulfate salts as an exemplar of the conditions thought to be associated with ancient Mars. We provide the first characterization of microbial structure in Spotted Lake sediments through metagenomic sequencing, and report a bacteria-dominated community with abundant Proteobacteria, Firmicutes, and Bacteroidetes, as well as diverse extremophiles. Microbial abundance and functional comparisons reveal similarities to Ace Lake, a meromictic Antarctic lake with anoxic and sulfidic bottom waters. Our analysis suggests that hypersaline-associated species occupy niches characterized foremost by differential abundance of Archaea, uncharacterized Bacteria, and Cyanobacteria. Potential biosignatures in this environment are discussed, specifically the likelihood of a strong sulfur isotopic fractionation record within the sediments due to the presence of sulfate reducing bacteria. With its high sulfate levels and seasonal freeze-thaw cycles, Spotted Lake is an analog for ancient paleolakes on Mars in which sulfate salt deposits may have offered periodically habitable environments, and could have concentrated and preserved organic materials or their biomarkers over geologic time.

Microbial Diversity of Impact-Generated Habitats.

Impact-generated lithologies have recently been identified as viable and important microbial habitats, especially within cold and arid regions such as the polar deserts on Earth. These unique habitats provide protection from environmental stressors, such as freeze-thaw events, desiccation, and UV radiation, and act to trap aerially deposited detritus within the fissures and pore spaces, providing necessary nutrients for endoliths. This study provides the first culture-independent analysis of the microbial community structure within impact-generated lithologies in a Mars analog environment, involving the analysis of 44,534 16S rRNA sequences from an assemblage of 21 rock samples that comprises three shock metamorphism categories. We find that species diversity increases (H = 2.4-4.6) with exposure to higher shock pressures, which leads to the development of three distinct populations. In each population, Actinobacteria were the most abundant (41%, 65%, and 59%), and the dominant phototrophic taxa came from the Chloroflexi. Calculated porosity (a function of shock metamorphism) for these samples correlates (R2 = 0.62) with inverse Simpson indices, accounting for overlap in populations in the higher shock levels. The results of our study show that microbial diversity is tied to the amount of porosity in the target substrate (as a function of shock metamorphism), resulting in the formation of distinct microbial populations. Key Words: Microbial diversity-Endoliths-Impact melt-rocks-Mars-Astrobiology. Astrobiology 16, 775-786.

Impact-generated endolithic habitat within crystalline rocks of the Haughton impact structure, Devon Island, Canada.

The colonization of rocks by endolithic communities is an advantageous trait, especially in environments such as hot or cold deserts, where large temperature ranges, low water availability, and high-intensity ultraviolet radiation pose a significant challenge to survival and growth. On Mars, similar conditions (albeit more extreme) prevail. In these environments, meteorite impact structures could provide refuge for endolithic organisms. Though initially detrimental to biology, an impact event into a rocky body can favorably change the availability and habitability of a substrate for endolithic organisms, which are then able to (re)colonize microfractures and pore spaces created during the impact. Here, we show how shocked gneisses from the Haughton impact structure, Devon Island, Canada, offer significant refuge for endolithic communities. A total of 28 gneiss samples representing a range of shock states were analyzed, collected from in situ, stable field locations. For each sample, the top centimeter of rock was examined with confocal scanning laser microscopy, scanning electron microscopy, and bright-field microscopy to investigate the relationship of biomass with shock level, which was found to correlate generally with increased shock state and particularly with increased porosity. We found that gneisses, which experienced pressures between 35 and 60 GPa, provide the most ideal habitat for endolithic organisms.