Experimental Identification of Potential Martian Biosignatures in Open and Closed Systems.

NASA's Perseverance and ESA's Rosalind Franklin rovers have the scientific goal of searching for evidence of ancient life on Mars. Geochemical biosignatures that form because of microbe-mineral interactions could play a key role in achieving this, as they can be preserved for millions of years on Earth, and the same could be true for Mars. Previous laboratory experiments have explored the formation of biosignatures under closed systems, but these do not represent the open systems that are found in natural martian environments, such as channels and lakes. In this study, we have conducted environmental simulation experiments using a global regolith simulant (OUCM-1), a thermochemically modelled groundwater, and an anaerobic microbial community to explore the formation of geochemical biosignatures within plausible open and closed systems on Mars. This initial investigation showed differences in the diversity of the microbial community developed after 28 days. In an open-system simulation (flow-through experiment), the acetogenic Acetobacterium (49% relative abundance) and the sulfate reducer Desulfosporomusa (43% relative abundance) were the dominant genera. Whereas in the batch experiment, the sulfate reducers Desulfovibrio, Desulfomicrobium, and Desulfuromonas (95% relative abundance in total) were dominant. We also found evidence of enhanced mineral dissolution within the flow-through experiment, but there was little evidence of secondary deposits in the presence of biota. In contrast, SiO2 and Fe deposits formed within the batch experiment with biota but not under abiotic conditions. The results from these initial experiments indicate that different geochemical biosignatures can be generated between open and closed systems, and therefore, biosignature formation in open systems warrants further investigation.

How to identify cell material in a single ice grain emitted from Enceladus or Europa.

Icy moons like Enceladus, and perhaps Europa, emit material sourced from their subsurface oceans into space via plumes of ice grains and gas. Both moons are prime targets for astrobiology investigations. Cassini measurements revealed a large compositional diversity of emitted ice grains with only 1 to 4% of Enceladus's plume ice grains containing organic material in high concentrations. Here, we report experiments simulating mass spectra of ice grains containing one bacterial cell, or fractions thereof, as encountered by advanced instruments on board future space missions to Enceladus or Europa, such as the SUrface Dust Analyzer onboard NASA's upcoming Europa Clipper mission at flyby speeds of 4 to 6 kilometers per second. Mass spectral signals characteristic of the bacteria are shown to be clearly identifiable by future missions, even if an ice grain contains much less than one cell. Our results demonstrate the advantage of analyses of individual ice grains compared to a diluted bulk sample in a heterogeneous plume.

Planetary Protection Knowledge Gap Closure Enabling Crewed Missions to Mars.

As focus for exploration of Mars transitions from current robotic explorers to development of crewed missions, it remains important to protect the integrity of scientific investigations at Mars, as well as protect the Earth's biosphere from any potential harmful effects from returned martian material. This is the discipline of planetary protection, and the Committee on Space Research (COSPAR) maintains the consensus international policy and guidelines on how this is implemented. Based on National Aeronautics and Space Administration (NASA) and European Space Agency (ESA) studies that began in 2001, COSPAR adopted principles and guidelines for human missions to Mars in 2008. At that point, it was clear that to move from those qualitative provisions, a great deal of work and interaction with spacecraft designers would be necessary to generate meaningful quantitative recommendations that could embody the intent of the Outer Space Treaty (Article IX) in the design of such missions. Beginning in 2016, COSPAR then sponsored a multiyear interdisciplinary meeting series to address planetary protection "knowledge gaps" (KGs) with the intent of adapting and extending the current robotic mission-focused Planetary Protection Policy to support the design and implementation of crewed and hybrid exploration missions. This article describes the outcome of the interdisciplinary COSPAR meeting series, to describe and address these KGs, as well as identify potential paths to gap closure. It includes the background scientific basis for each topic area and knowledge updates since the meeting series ended. In particular, credible solutions for KG closure are described for the three topic areas of (1) microbial monitoring of spacecraft and crew health; (2) natural transport (and survival) of terrestrial microbial contamination at Mars, and (3) the technology and operation of spacecraft systems for contamination control. The article includes a KG data table on these topic areas, which is intended to be a point of departure for making future progress in developing an end-to-end planetary protection requirements implementation solution for a crewed mission to Mars. Overall, the workshop series has provided evidence of the feasibility of planetary protection implementation for a crewed Mars mission, given (1) the establishment of needed zoning, emission, transport, and survival parameters for terrestrial biological contamination and (2) the creation of an accepted risk-based compliance approach for adoption by spacefaring actors including national space agencies and commercial/nongovernment organizations.

Draft genome sequence of Bacillus sp. strain X and Salarachaeum sp. strain III isolated from Lake Karum, Danakil Depression, Ethiopia.

Here, we report the draft genome sequences of strains of Bacillus and Salarachaeum that were isolated from hypersaline water samples collected from Lake Karum, Danakil Depression, Ethiopia. The sequences pave the way for more targeted studies into the potential biological activities and secondary metabolite synthesis of these organisms.

Draft genome sequence of Halobacillus campisalis strain ASL-17.

We report here the genome sequence of moderately halophilic Halobacillus campisalis ASL-17, isolated from hypersaline sediment from the Yellow Sea, Korea. The bacterium was Gram variable, oval or coccoid, and mesophilic. The genome of H. campisalis ASL-17 has 3.8 Mbp, with 3,910 coding sequences, 76 RNAs, and 41.3% G + C content.

Diversity of Microbial Mats in the Makgadikgadi Salt Pans, Botswana.

The Makgadikgadi Salt Pans are the remnants of a mega paleo-lake system in the central Kalahari, Botswana. Today, the Makgadikgadi Basin is an arid to semi-arid area receiving water of meteoric origin during the short, wet season. Large microbial mats, which support primary production, are formed due to desiccation during the dry season. This study aimed to characterise the microbial diversity of the microbial mats and the underlying sediment. The focus was the Ntwetwe Pan, located west of the Makgadikgadi Basin. Metagenomic analyses demonstrated that the mats consisted of a high relative abundance of Cyanobacteriota (synonym Cyanobacteria) (20.50-41.47%), Pseudomonadota (synonym Proteobacteria) (15.71 to 32.18%), and Actinomycetota (synonym Actinobacteria) (8.53-32.56%). In the underlying sediments, Pseudomonadota, Actinomycetota, and Euryarchaeota represented over 70% of the community. Localised fluctuations in water content and pH did not significantly affect the microbial diversity of the sediment or the mats.

The COSPAR planetary protection requirements for space missions to Venus.

The Committee on Space Research's (COSPAR) Planetary Protection Policy states that all types of missions to Venus are classified as Category II, as the planet has significant research interest relative to the processes of chemical evolution and the origin of life, but there is only a remote chance that terrestrial contamination can proliferate and compromise future investigations. "Remote chance" essentially implies the absence of environments where terrestrial organisms could survive and replicate. Hence, Category II missions only require simplified planetary protection documentation, including a planetary protection plan that outlines the intended or potential impact targets, brief Pre- and Post-launch analyses detailing impact strategies, and a Post-encounter and End-of-Mission Report. These requirements were applied in previous missions and are foreseen for the numerous new international missions planned for the exploration of Venus, which include NASA's VERITAS and DAVINCI missions, and ESA's EnVision mission. There are also several proposed missions including India's Shukrayaan-1, and Russia's Venera-D. These multiple plans for spacecraft coincide with a recent interest within the scientific community regarding the cloud layers of Venus, which have been suggested by some to be habitable environments. The proposed, privately funded, MIT/Rocket Lab Venus Life Finder mission is specifically designed to assess the habitability of the Venusian clouds and to search for signs of life. It includes up to three atmospheric probes, the first one targeting a launch in 2023. The COSPAR Panel on Planetary Protection evaluated scientific data that underpins the planetary protection requirements for Venus and the implications of this on the current policy. The Panel has done a thorough review of the current knowledge of the planet's conditions prevailing in the clouds. Based on the existing literature, we conclude that the environmental conditions within the Venusian clouds are orders of magnitude drier and more acidic than the tolerated survival limits of any known terrestrial extremophile organism. Because of this future orbital, landed or entry probe missions to Venus do not require extra planetary protection measures. This recommendation may be revised in the future if new observations or reanalysis of past data show any significant increment, of orders of magnitude, in the water content and the pH of the cloud layer.

The COSPAR Planetary Protection Policy for robotic missions to Mars: A review of current scientific knowledge and future perspectives.

Planetary protection guidance for martian exploration has become a notable point of discussion over the last decade. This is due to increased scientific interest in the habitability of the red planet with updated techniques, missions becoming more attainable by smaller space agencies, and both the private sector and governments engaging in activities to facilitate commercial opportunities and human-crewed missions. The international standards for planetary protection have been developed through consultation with the scientific community and the space agencies by the Committee on Space Research's (COSPAR) Panel on Planetary Protection, which provides guidance for compliance with the Outer Space Treaty of 1967. In 2021, the Panel evaluated recent scientific data and literature regarding the planetary protection requirements for Mars and the implications of this on the guidelines. In this paper, we discuss the COSPAR Planetary Protection Policy for Mars, review the new scientific findings and discuss the next steps required to enable the next generation of robotic missions to Mars.

Habitability and Biosignature Formation in Simulated Martian Aqueous Environments.

Water present on early Mars is often assumed to have been habitable. In this study, experiments were performed to investigate the habitability of well-defined putative martian fluids and to identify the accompanying potential formation of biosignatures. Simulated martian environments were developed by combining martian fluid and regolith simulants based on the chemistry of the Rocknest sand shadow at Gale Crater. The simulated chemical environment was inoculated with terrestrial anoxic sediment from the Pyefleet mudflats (United Kingdom). These enrichments were cultured for 28 days and subsequently subcultured seven times to ensure that the microbial community was solely grown on the defined, simulated chemistry. The impact of the simulated chemistries on the microbial community was assessed by cell counts and sequencing of 16S rRNA gene profiles. Associated changes to the fluid and precipitate chemistries were established by using ICP-OES, IC, FTIR, and NIR. The fluids were confirmed as habitable, with the enriched microbial community showing a reduction in abundance and diversity over multiple subcultures relating to the selection of specific metabolic groups. The final community comprised sulfate-reducing, acetogenic, and other anaerobic and fermentative bacteria. Geochemical characterization and modeling of the simulant and fluid chemistries identified clear differences between the biotic and abiotic experiments. These differences included the elimination of sulfur owing to the presence of sulfate-reducing bacteria and more general changes in pH associated with actively respiring cells that impacted the mineral assemblages formed. This study confirmed that a system simulating the fluid chemistry of Gale Crater could support a microbial community and that variation in chemistries under biotic and abiotic conditions can be used to inform future life-detection missions.

Toward Detecting Biosignatures of DNA, Lipids, and Metabolic Intermediates from Bacteria in Ice Grains Emitted by Enceladus and Europa.

The reliable identification of biosignatures is key to the search for life elsewhere. On ocean worlds like Enceladus or Europa, this can be achieved by impact ionization mass spectrometers, such as the SUrface Dust Analyzer (SUDA) on board NASA's upcoming Europa Clipper mission. During spacecraft flybys, these instruments can sample ice grains formed from subsurface water and emitted by these moons. Previous laboratory analog experiments have demonstrated that SUDA-type instruments could identify amino acids, fatty acids, and peptides in ice grains and discriminate between their abiotic and biotic origins. Here, we report experiments simulating impact ionization mass spectra of ice grains containing DNA, lipids, and metabolic intermediates extracted from two bacterial cultures: Escherichia coli and Sphingopyxis alaskensis. Salty Enceladan or Europan ocean waters were simulated using matrices with different NaCl concentrations. Characteristic mass spectral signals, such as DNA nucleobases, are clearly identifiable at part-per-million-level concentrations. Mass spectra of all substances exhibit unambiguous biogenic patterns, which in some cases show significant differences between the two bacterial species. Sensitivity to the biosignatures decreases with increasing matrix salinity. The experimental parameters indicate that future impact ionization mass spectrometers will be most sensitive to the investigated biosignatures for ice grain encounter speeds of 4-6 km/s.

Biosignature stability in space enables their use for life detection on Mars.

Two rover missions to Mars aim to detect biomolecules as a sign of extinct or extant life with, among other instruments, Raman spectrometers. However, there are many unknowns about the stability of Raman-detectable biomolecules in the martian environment, clouding the interpretation of the results. To quantify Raman-detectable biomolecule stability, we exposed seven biomolecules for 469 days to a simulated martian environment outside the International Space Station. Ultraviolet radiation (UVR) strongly changed the Raman spectra signals, but only minor change was observed when samples were shielded from UVR. These findings provide support for Mars mission operations searching for biosignatures in the subsurface. This experiment demonstrates the detectability of biomolecules by Raman spectroscopy in Mars regolith analogs after space exposure and lays the groundwork for a consolidated space-proven database of spectroscopy biosignatures in targeted environments.

Oligotrophic Growth of Nitrate-Dependent Fe2+-Oxidising Microorganisms Under Simulated Early Martian Conditions.

Nitrate-dependent Fe2+ oxidation (NDFO) is a microbially mediated process observed in many anaerobic, low-nutrient (oligotrophic) neutral-alkaline environments on Earth, which describes oxidation of Fe2+ to Fe3+ in tandem with microbial nitrate reduction. Evidence suggests that similar environments existed on Mars during the Noachian epoch (4.1-3.7 Ga) and in periodic, localised environments more recently, indicating that NDFO metabolism could have played a role in a potential early martian biosphere. In this paper, three NDFO microorganisms, Acidovorax sp. strain BoFeN1, Pseudogulbenkiania sp. strain 2002 and Paracoccus sp. strain KS1, were assessed for their ability to grow oligotrophically in simulated martian brines and in a minimal medium with olivine as a solid Fe2+ source. These simulant-derived media were developed from modelled fluids based on the geochemistry of Mars sample locations at Rocknest (contemporary Mars soil), Paso Robles (sulphur-rich soil), Haematite Slope (haematite-rich soil) and a Shergottite meteorite (common basalt). The Shergottite medium was able to support growth of all three organisms, while the contemporary Mars medium supported growth of Acidovorax sp. strain BoFeN1 and Pseudogulbenkiania sp. strain 2002; however, growth was not accompanied by significant Fe2+ oxidation. Each of the strains was also able to grow in oligotrophic minimal media with olivine as the sole Fe2+ source. Biomineralised cells of Pseudogulbenkiania sp. strain 2002 were identified on the surface of the olivine, representing a potential biosignature for NDFO microorganisms in martian samples. The results suggest that NDFO microorganisms could have thrived in early martian groundwaters under oligotrophic conditions, depending on the local lithology. This can guide missions in identifying palaeoenvironments of interest for biosignature detection. Indeed, biomineralised cells identified on the olivine surface provide a previously unexplored mechanism for the preservation of morphological biosignatures in the martian geological record.

Assembly of Bacterial Genome Sequences from Metagenomes of Spacecraft Assembly Cleanrooms.

Characterizing the microbiome of spacecraft assembly cleanrooms is important for planetary protection. We report two bacterial metagenome-assembled genomes (MAGs) reconstructed from metagenomes produced from cleanroom samples from the Kennedy Space Center's Payload Hazardous Servicing Facility (KSC-PHSF) during the handling of the Phoenix spacecraft. Characterization of these MAGs will enable identification of the strategies underpinning their survival.

Habitability of Hydrothermal Systems at Jezero and Gusev Craters as Constrained by Hydrothermal Alteration of a Terrestrial Mafic Dike.

NASA's search for habitable environments has focused on alteration mineralogy of the Martian crust and the formation of hydrous minerals, because they reveal information about the fluid and environmental conditions from which they precipitated. Extensive work has focused on the formation of alteration minerals at low temperatures, with limited work investigating metamorphic or high-temperature alteration. We have investigated such a site as an analog for Mars: a mafic dike on the Colorado Plateau that was hydrothermally altered from contact with groundwater as it was emplaced in the porous and permeable Jurassic Entrada sandstone. Our results show evidence for fluid mobility removing Si and K but adding S, Fe, Ca, and possibly Mg to the system as alteration progresses. Mineralogically, all samples contain calcite, hematite, and kaolinite; with most samples containing minor anatase, barite, halite, and dolomite. The number of alteration minerals increase with alteration. The hydrothermal system that formed during interaction of the magma (heat source) and groundwater would have been a habitable environment once the system cooled below ~120° C. The mineral assemblage is similar to alteration minerals seen within the Martian crust from orbit, including those at Gusev and Jezero Craters. Therefore, based on our findings, and extrapolating them to the Martian crust, these sites may represent habitable environments which would call for further exploration and sample return of such hydrothermally altered igneous materials.

Author Correction: Ultra-small microorganisms in the polyextreme conditions of the Dallol volcano, Northern Afar, Ethiopia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Experimental and Simulation Efforts in the Astrobiological Exploration of Exooceans.

The icy satellites of Jupiter and Saturn are perhaps the most promising places in the Solar System regarding habitability. However, the potential habitable environments are hidden underneath km-thick ice shells. The discovery of Enceladus' plume by the Cassini mission has provided vital clues in our understanding of the processes occurring within the interior of exooceans. To interpret these data and to help configure instruments for future missions, controlled laboratory experiments and simulations are needed. This review aims to bring together studies and experimental designs from various scientific fields currently investigating the icy moons, including planetary sciences, chemistry, (micro-)biology, geology, glaciology, etc. This chapter provides an overview of successful in situ, in silico, and in vitro experiments, which explore different regions of interest on icy moons, i.e. a potential plume, surface, icy shell, water and brines, hydrothermal vents, and the rocky core.

Exploring Deep-Sea Brines as Potential Terrestrial Analogues of Oceans in the Icy Moons of the Outer Solar System.

Several icy moons of the outer solar system have been receiving considerable attention and are currently seen as major targets for astrobiological research and the search for life beyond our planet. Despite the limited amount of data on the oceans of these moon, we expect them to be composed of brines with variable chemistry, some degree of hydrothermal input, and be under high pressure conditions. The combination of these different conditions significantly limits the number of extreme locations, which can be used as terrestrial analogues. Here we propose the use of deep-sea brines as potential terrestrial analogues to the oceans in the outer solar system. We provide an overview of what is currently known about the conditions on the icy moons of the outer solar system and their oceans as well as on deep-sea brines of the Red Sea and the Mediterranean and their microbiology. We also identify several threads of future research, which would be particularly useful in the context of future exploration of these extra-terrestrial oceans.

Infrared Spectroscopic Detection of Biosignatures at Lake Tírez, Spain: Implications for Mars.

The detection of potential biosignatures with mineral matrices is part of a multifaceted approach in the search for life on other planetary bodies. The 2020 ExoMars Rosalind Franklin rover includes within its payload three IR spectrometers in the form of ISEM (Infrared Spectrometer for ExoMars), MicrOmega, and Ma-MISS (Mars Multispectral Imager for Subsurface Studies). The use of this technique in the detection and characterization of biosignatures is of great value. Organic materials are often co-deposited in terrestrial evaporites and as such have been proposed as relevant analogs in the search for life on Mars. This study focuses on Ca-sulfates collected from the hypersaline Tírez Lake in Spain. Mid infrared and visible near infrared analysis of soils, salt crusts, and crystals with green and red layering indicative of microbial colonization of the samples was acquired from across the lake and identified the main mineral to be gypsum with inputs of carbonate and silica. Organic functional groups that could be attributed to amides and carboxylic acids were identified as well as chlorophyll; however, due to the strong mineralogical absorptions observed, these were hard to unambiguously discern. Taxonomical assignment demonstrated that the archaeal community within the samples was dominated by the halophilic extremophile Halobacteriaceae while the bacterial community was dominated by the class Nocardiaceae. The results of this research highlight that sulfates on Mars are a mixed blessing, acting as an effective host for organic matter preservation but also a material that masks the presence of organic functional groups when analyzed with spectroscopic tools similar to those due to fly on the 2020 ExoMars rover. A suite of complementary analytical techniques therefore should be used to support the spectral identification of any candidate extraterrestrial biosignatures.

Ultra-small microorganisms in the polyextreme conditions of the Dallol volcano, Northern Afar, Ethiopia.

The Dallol geothermal area in the northern part of the Danakil Depression (up to 124-155 meter below sea level) is deemed one of the most extreme environments on Earth. The area is notable for being part of the Afar Depression, an incipient seafloor-spreading center located at the triple junction, between Nubian, Somali and Arabian plates, and for hosting environments at the very edge of natural physical-chemical extremities. The northern part of the Danakil Depression is dominated by the Assale salt plain (an accumulation of marine evaporite deposits) and hosts the Dallol volcano. Here, the interaction between the evaporitic deposit and the volcanisms have created the unique Dallol hot springs, which are highly acidic (pH ~ 0) and saline (saturation) with maximum temperatures ranging between 90 and 109 °C. Here we report for the first time evidence of life existing with these hot springs using a combination of morphological and molecular analyses. Ultra-small structures are shown to be entombed within mineral deposits, which are identified as members of the Order Nanohaloarchaea. The results from this study suggest the microorganisms can survive, and potential live, within this extreme environment, which has implications for understanding the limits of habitability on Earth and on (early) Mars.

Carbon Fixation by Marine Ultrasmall Prokaryotes.

Autotrophic carbon fixation is a crucial process for sustaining life on Earth. To date, six pathways, the Calvin-Benson-Bassham cycle, the reductive tricarboxylic acid cycle, the 3-hydroxypropionate bi-cycle, the Wood-Ljungdahl pathway, the dicarboxylate/4-hydroxybutyrate cycle, and the 4-hydroxybutyrate cycle, have been described. Nano-organisms such as members of the Candidate Phyla Radiation (CPR) bacterial superphylum and the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohalorchaeota (DPANN) archaeal superphylum could deeply impact carbon cycling and carbon fixation in ways that are still to be determined. CPR and DPANN are ubiquitous in the environment but understudied; their gene contents are not exhaustively described; and their metabolisms are not yet fully understood. Here, the completeness of each of the above pathways was quantified and tested for the presence of all key enzymes in nano-organisms from across the World Ocean. The novel marine ultrasmall prokaryotes were demonstrated to collectively harbor the genes required for carbon fixation, in particular the "energetically efficient" dicarboxylate/4-hydroxybutyrate pathway and the 4-hydroxybutyrate pathway. This contrasted with the known carbon metabolic pathways associated with CPR members in aquifers, where they are described as degraders (Castelle CJ, et al. 2015. Genomic expansion of domain archaea highlights roles for organisms from new phyla in anaerobic carbon cycling. Curr Biol. 25(6):690-701; Castelle CJ, et al. 2018. Biosynthetic capacity, metabolic variety and unusual biology in the CPR and DPANN radiations. Nat Rev Microbiol. 16(10):629-645; Anantharaman K, et al. 2016. Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system. Nat Commun. 7:13219.). Our findings suggest that nano-organisms have a broader contribution to carbon fixation and cycling than currently assumed. Furthermore, CPR and DPANN superphyla are possibly not the only nanosized prokaryotes; therefore, the discovery of new autotrophic marine nano-organisms by future single cell genomics is anticipated.