Investigating Microbial Biosignatures in Aeolian Environments Using Micro-X-Ray: Simulation of PIXL Instrument Analyses at Jezero Crater Onboard the Perseverance Mars 2020 Rover.

Assessing the past habitability of Mars and searching for evidence of ancient life at Jezero crater via the Perseverance rover are the key objectives of NASA's Mars 2020 mission. Onboard the rover, PIXL (Planetary Instrument for X-ray Lithochemistry) is one of the best suited instruments to search for microbial biosignatures due to its ability to characterize chemical composition of fine scale textures in geological targets using a nondestructive technique. PIXL is also the first micro-X-ray fluorescence (XRF) spectrometer onboard a Mars rover. Here, we present guidelines for identifying and investigating a microbial biosignature in an aeolian environment using PIXL-analogous micro-XRF (µXRF) analyses. We collected samples from a modern wet aeolian environment at Padre Island, Texas, that contain buried microbial mats, and we analyzed them using µXRF techniques analogous to how PIXL is being operated on Mars. We show via µXRF technique and microscope images the geochemical and textural variations from the surface to ∼40 cm depth. Microbial mats are associated with heavy-mineral lags and show specific textural and geochemical characteristics that make them a distinct biosignature for this environment. Upon burial, they acquire a diffuse texture due to the expansion and contraction of gas-filled voids, and they present a geochemical signature rich in iron and titanium, which is due to the trapping of heavy minerals. We show that these intrinsic characteristics can be detected via µXRF analyses, and that they are distinct from buried abiotic facies such as cross-stratification and adhesion ripple laminations. We also designed and conducted an interactive survey using the Padre Island µXRF data to explore how different users chose to investigate a biosignature-bearing dataset via PIXL-like sampling strategies. We show that investigating biosignatures via PIXL-like analyses is heavily influenced by technical constraints (e.g., the XRF measurement characteristics) and by the variety of approaches chosen by different scientists. Lessons learned for accurately identifying and characterizing this biosignature in the context of rover-mission constraints include defining relative priorities among measurements, favoring a multidisciplinary approach to the decision-making process of XRF measurements selection, and considering abiotic results to support or discard a biosignature interpretation. Our results provide guidelines for PIXL analyses of potential biosignature on Mars.

LOUPE: observing Earth from the Moon to prepare for detecting life on Earth-like exoplanets.

LOUPE, the Lunar Observatory for Unresolved Polarimetry of the Earth, is a small, robust spectro-polarimeter for observing the Earth as an exoplanet. Detecting Earth-like planets in stellar habitable zones is one of the key challenges of modern exoplanetary science. Characterizing such planets and searching for traces of life requires the direct detection of their signals. LOUPE provides unique spectral flux and polarization data of sunlight reflected by Earth, the only planet known to harbour life. These data will be used to test numerical codes to predict signals of Earth-like exoplanets, to test algorithms that retrieve planet properties, and to fine-tune the design and observational strategies of future space observatories. From the Moon, LOUPE will continuously see the entire Earth, enabling it to monitor the signal changes due to the planet's daily rotation, weather patterns and seasons, across all phase angles. Here, we present both the science case and the technology behind LOUPE's instrumental and mission design. This article is part of a discussion meeting issue 'Astronomy from the Moon: the next decades'.

Investigating Habitability with an Integrated Rock-Climbing Robot and Astrobiology Instrument Suite.

A prototype rover carrying an astrobiology payload was developed and deployed at analog field sites to mature generalized system architectures capable of searching for biosignatures in extreme terrain across the Solar System. Specifically, the four-legged Limbed Excursion Mechanical Utility Robot (LEMUR) 3 climbing robot with microspine grippers carried three instruments: a micro-X-ray fluorescence instrument based on the Mars 2020 mission's Planetary Instrument for X-ray Lithochemistry provided elemental chemistry; a deep-ultraviolet fluorescence instrument based on Mars 2020's Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals mapped organics in bacterial communities on opaque substrates; and a near-infrared acousto-optic tunable filter-based point spectrometer identified minerals and organics in the 1.6-3.6 µm range. The rover also carried a light detection and ranging and a color camera for both science and navigation. Combined, this payload detects astrobiologically important classes of rock components (elements, minerals, and organics) in extreme terrain, which, as demonstrated in this work, can reveal a correlation between textural biosignatures and the organics or elements expected to preserve them in a habitable environment. Across >10 field tests, milestones were achieved in instrument operations, autonomous mobility in extreme terrain, and system integration that can inform future planetary science mission architectures. Contributions include (1) system-level demonstration of mock missions to the vertical exposures of Mars lava tube caves and Mars canyon walls, (2) demonstration of multi-instrument integration into a confocal arrangement with surface scanning capabilities, and (3) demonstration of automated focus stacking algorithms for improved signal-to-noise ratios and reduced operation time.

The Complex Molecules Detector (CMOLD): A Fluidic-Based Instrument Suite to Search for (Bio)chemical Complexity on Mars and Icy Moons.

Organic chemistry is ubiquitous in the Solar System, and both Mars and a number of icy satellites of the outer Solar System show substantial promise for having hosted or hosting life. Here, we propose a novel astrobiologically focused instrument suite that could be included as scientific payload in future missions to Mars or the icy moons: the Complex Molecules Detector, or CMOLD. CMOLD is devoted to determining different levels of prebiotic/biotic chemical and structural targets following a chemically general approach (i.e., valid for both terrestrial and nonterrestrial life), as well as their compatibility with terrestrial life. CMOLD is based on a microfluidic block that distributes a liquid suspension sample to three instruments by using complementary technologies: (1) novel microscopic techniques for identifying ultrastructures and cell-like morphologies, (2) Raman spectroscopy for detecting universal intramolecular complexity that leads to biochemical functionality, and (3) bioaffinity-based systems (including antibodies and aptamers as capture probes) for finding life-related and nonlife-related molecular structures. We highlight our current developments to make this type of instruments flight-ready for upcoming Mars missions: the Raman spectrometer included in the science payload of the ESAs Rosalind Franklin rover (Raman Laser Spectrometer instrument) to be launched in 2022, and the biomarker detector that was included as payload in the NASA Icebreaker lander mission proposal (SOLID instrument). CMOLD is a robust solution that builds on the combination of three complementary, existing techniques to cover a wide spectrum of targets in the search for (bio)chemical complexity in the Solar System.

Molecular Muscle Experiment: Hardware and Operational Lessons for Future Astrobiology Space Experiments.

Biology experiments in space seek to increase our understanding of what happens to life beyond Earth and how we can safely send life beyond Earth. Spaceflight is associated with many (mal)adaptations in physiology, including decline in musculoskeletal, cardiovascular, vestibular, and immune systems. Biological experiments in space are inherently challenging to implement. Development of hardware and validation of experimental conditions are critical to ensure the collection of high-quality data. The model organism Caenorhabditis elegans has been studied in space for more than 20 years to better understand spaceflight-induced (patho)physiology, particularly spaceflight-induced muscle decline. These experiments have used a variety of hardware configurations. Despite this, hardware used in the past was not available for our most recent experiment, the Molecular Muscle Experiment (MME). Therefore, we had to design and validate flight hardware for MME. MME provides a contemporary example of many of the challenges faced by researchers conducting C. elegans experiments onboard the International Space Station. Here, we describe the hardware selection and validation, in addition to the ground-based experiment scientific validation testing. These experiences and operational solutions allow others to replicate and/or improve our experimental design on future missions.

Exo-Ocean Exploration with Deep-Sea Sensor and Platform Technologies.

One of Saturn's largest moons, Enceladus, possesses a vast extraterrestrial ocean (i.e., exo-ocean) that is increasingly becoming the hotspot of future research initiatives dedicated to the exploration of putative life. Here, a new bio-exploration concept design for Enceladus' exo-ocean is proposed, focusing on the potential presence of organisms across a wide range of sizes (i.e., from uni- to multicellular and animal-like), according to state-of-the-art sensor and robotic platform technologies used in terrestrial deep-sea research. In particular, we focus on combined direct and indirect life-detection capabilities, based on optoacoustic imaging and passive acoustics, as well as molecular approaches. Such biologically oriented sampling can be accompanied by concomitant geochemical and oceanographic measurements to provide data relevant to exo-ocean exploration and understanding. Finally, we describe how this multidisciplinary monitoring approach is currently enabled in terrestrial oceans through cabled (fixed) observatories and their related mobile multiparametric platforms (i.e., Autonomous Underwater and Remotely Operated Vehicles, as well as crawlers, rovers, and biomimetic robots) and how their modified design can be used for exo-ocean exploration.

Is a Linear or a Walkabout Protocol More Efficient When Using a Rover to Choose Biologically Relevant Samples in a Small Region of Interest?

We conducted a field test at a potential Mars analog site to provide insight into planning for future robotic missions such as Mars 2020, where science operations must facilitate efficient choice of biologically relevant sampling locations. We compared two data acquisition and decision-making protocols currently used by Mars Science Laboratory: (1) a linear approach, where sites are examined as they are encountered and (2) a walkabout approach, in which the field site is first examined with remote rover instruments to gain an understanding of regional context followed by deployment of time- and power-intensive contact and sampling instruments on a smaller subset of locations. The walkabout method was advantageous in terms of both the time required to execute and a greater confidence in results and interpretations, leading to enhanced ability to tailor follow-on observations to better address key science and sampling goals. This advantage is directly linked to the walkabout method's ability to provide broad geological context earlier in the science analysis process. For Mars 2020, and specifically for small regions to be explored (e.g., <1 km2), we recommend that the walkabout approach be considered where possible, to provide early context and time for the science team to develop a coherent suite of hypotheses and robust ways to test them.

Detection of Potential Lipid Biomarkers in Oxidative Environments by Raman Spectroscopy and Implications for the ExoMars 2020-Raman Laser Spectrometer Instrument Performance.

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).

ExoMars Raman Laser Spectrometer: A Tool for the Potential Recognition of Wet-Target Craters on Mars.

In the present work, near-infrared, laser-induced breakdown spectroscopy, Raman, and X-ray diffractometer techniques have been complementarily used to carry out a comprehensive characterization of a terrestrial analogue selected from the Chesapeake Bay impact structure (CBIS). The obtained data clearly highlight the key role of Raman spectroscopy in the detection of minor and trace compounds, through which inferences about geological processes occurred in the CBIS can be extrapolated. Beside the use of commercial systems, further Raman analyses were performed by the Raman laser spectrometer (RLS) ExoMars Simulator. This instrument represents the most reliable tool to effectively predict the scientific capabilities of the ExoMars/Raman system that will be deployed on Mars in 2021. By emulating the analytical procedures and operational restrictions established by the ExoMars mission rover design, it was proved that the RLS ExoMars Simulator can detect the amorphization of quartz, which constitutes an analytical clue of the impact origin of craters. Beside amorphized minerals, the detection of barite and siderite, compounds crystallizing under hydrothermal conditions, helps indirectly to confirm the presence of water in impact targets. Furthermore, the RLS ExoMars Simulator capability of performing smart molecular mappings was successfully evaluated.

Testing Flight-like Pyrolysis Gas Chromatography-Mass Spectrometry as Performed by the Mars Organic Molecule Analyzer Onboard the ExoMars 2020 Rover on Oxia Planum Analog Samples.

The Mars Organic Molecule Analyzer (MOMA) onboard the ExoMars 2020 rover (to be landed in March 2021) utilizes pyrolysis gas chromatography-mass spectrometry (GC-MS) with the aim to detect organic molecules in martian (sub-) surface materials. Pyrolysis, however, may thermally destroy and transform organic matter depending on the temperature and nature of the molecules, thus altering the original molecular signatures. In this study, we tested MOMA flight-like pyrolysis GC-MS without the addition of perchlorates on well-characterized natural mineralogical analog samples for Oxia Planum, the designated ExoMars 2020 landing site. Experiments were performed on an iron-rich shale (that is rich in Fe-Mg-smectites) and an opaline chert, with known organic matter compositions, to test pyrolytic effects related to heating in the MOMA oven. Two hydrocarbon standards (n-octadecane and phytane) were also analyzed. The experiments show that during stepwise pyrolysis (300°C, 500°C, and 700°C), (1) low-molecular-weight hydrocarbon biomarkers (such as acyclic isoprenoids and aryl isoprenoids) can be analyzed intact, (2) discrimination between free and complex molecules (macromolecules) is principally possible, (3) secondary pyrolysis products and carryover may affect the 500°C and 700°C runs, and (4) the type of the organic matter (functionalized vs. defunctionalized) governs the pyrolysis outcome rather than the difference in mineralogy. Although pyrosynthesis reactions and carryover clearly have to be considered in data interpretation, our results demonstrate that pyrolysis GC-MS onboard MOMA operated under favorable conditions (e.g., no perchlorates) will be capable of providing important structural information on organic matter found on Mars, particularly when used in conjunction with other techniques on MOMA, including derivatization and thermochemolysis GC-MS and laser desorption/ionization-MS.

Phosphine as a Biosignature Gas in Exoplanet Atmospheres.

A long-term goal of exoplanet studies is the identification and detection of biosignature gases. Beyond the most discussed biosignature gas O2, only a handful of gases have been considered in detail. In this study, we evaluate phosphine (PH3). On Earth, PH3 is associated with anaerobic ecosystems, and as such, it is a potential biosignature gas in anoxic exoplanets. We simulate the atmospheres of habitable terrestrial planets with CO2- and H2-dominated atmospheres and find that PH3 can accumulate to detectable concentrations on planets with surface production fluxes of 1010 to 1014 cm-2 s-1 (corresponding to surface concentrations of 10s of ppb to 100s of ppm), depending on atmospheric composition and ultraviolet (UV) irradiation. While high, the surface flux values are comparable to the global terrestrial production rate of methane or CH4 (1011 cm-2 s-1) and below the maximum local terrestrial PH3 production rate (1014 cm-2 s-1). As with other gases, PH3 can more readily accumulate on low-UV planets, for example, planets orbiting quiet M dwarfs or with a photochemically generated UV shield. PH3 has three strong spectral features such that in any atmosphere scenario one of the three will be unique compared with other dominant spectroscopic molecules. Phosphine's weakness as a biosignature gas is its high reactivity, requiring high outgassing rates for detectability. We calculate that tens of hours of JWST (James Webb Space Telescope) time are required for a potential detection of PH3. Yet, because PH3 is spectrally active in the same wavelength regions as other atmospherically important molecules (such as H2O and CH4), searches for PH3 can be carried out at no additional observational cost to searches for other molecular species relevant to characterizing exoplanet habitability. Phosphine is a promising biosignature gas, as it has no known abiotic false positives on terrestrial planets from any source that could generate the high fluxes required for detection.

Investigating the Effect of Perchlorate on Flight-like Gas Chromatography-Mass Spectrometry as Performed by MOMA on board the ExoMars 2020 Rover.

The Mars Organic Molecule Analyzer (MOMA) instrument on board ESA's ExoMars 2020 rover will be essential in the search for organic matter. MOMA applies gas chromatography-mass spectrometry (GC-MS) techniques that rely on thermal volatilization. Problematically, perchlorates and chlorates in martian soils and rocks become highly reactive during heating (>200°C) and can lead to oxidation and chlorination of organic compounds, potentially rendering them unidentifiable. Here, we analyzed a synthetic sample (alkanols and alkanoic acids on silica gel) and a Silurian chert with and without Mg-perchlorate to evaluate the applicability of MOMA-like GC-MS techniques to different sample types and assess the impact of perchlorate. We used a MOMA flight analog system coupled to a commercial GC-MS to perform MOMA-like pyrolysis, in situ derivatization, and in situ thermochemolysis. We show that pyrolysis can provide a sufficient overview of the organic inventory but is strongly affected by the presence of perchlorates. In situ derivatization facilitates the identification of functionalized organics but showed low efficiency for n-alkanoic acids. Thermochemolysis is shown to be an effective technique for the identification of both refractory and functional compounds. Most importantly, this technique was barely affected by perchlorates. Therefore, MOMA GC-MS analyses of martian surface/subsurface material may be less affected by perchlorates than commonly thought, in particular when applying the full range of available MOMA GC-MS techniques.

Gene Expression Measurement Module (GEMM) for space application: Design and validation.

In order to facilitate studies on the impact of the space environment on biological systems, we have developed a prototype of GEMM (Gene Expression Measurement Module) - an automated, miniaturized, integrated fluidic system for in-situ measurements of gene expression in microbial samples. The GEMM instrument is capable of (1) lysing bacterial cell walls, (2) extracting and purifying RNA released from cells, (3) hybridizing the RNA to probes attached to a microarray and (4) providing electrochemical readout, all in a microfluidics cartridge. To function on small, uncrewed spacecraft, the conventional, laboratory protocols for both sample preparation and hybridization required significant modifications. Biological validation of the instrument was carried out on Synechococcus elongatus, a photosynthetic cyanobacterium known for its metabolic diversity and resilience to adverse conditions. It was demonstrated that GEMM yielded reliable, reproducible gene expression profiles. GEMM is the only high throughput instrument that can be deployed in near future on space platforms other than the ISS to advance biological research in space. It can also prove useful for numerous terrestrial applications in the field.

In Situ Geochronology on Mars and the Development of Future Instrumentation.

We review the in situ geochronology experiments conducted by the Mars Science Laboratory mission's Curiosity rover to understand when the Gale Crater rocks formed, underwent alteration, and became exposed to cosmogenic radiation. These experiments determined that the detrital minerals in the sedimentary rocks of Gale are ∼4 Ga, consistent with their origin in the basalts surrounding the crater. The sedimentary rocks underwent fluid-moderated alteration 2 Gyr later, which may mark the closure of aqueous activity at Gale Crater. Over the past several million years, wind-driven processes have dominated, denuding the surfaces by scarp retreat. The Curiosity measurements validate radiometric dating techniques on Mars and guide the way for future instrumentation to make more precise measurements that will further our understanding of the geological and astrobiological history of the planet.

A Simple Instrument Suite for Characterizing Habitability and Weathering: The Modern Aqueous Habitat Reconnaissance Suite (MAHRS).

The shallow subsurface of Mars is extremely interesting as a possible microbial habitat because it becomes temporarily wet, it is shielded from radiation, and mixing by aeolian processes could provide the sources of energy and nutrients necessary for sustaining microbial life in it. The Modern Aqueous Habitat Reconnaissance Suite (MAHRS) was developed primarily to search for potentially habitable environments in the shallow subsurface of Mars and to study weathering, but it can also be used to search for potentially habitable environments in the shallow subsurface of other planetary bodies such as the Icy Worlds. MAHRS includes an instrument developed to measure regolith wetness and search for brine in the shallow subsurface of Mars, where it is most likely to be found. The detection of brine can aid in our understanding not only of habitability but also of geochemistry and aqueous weathering processes. Besides the regolith wetness sensor, MAHRS includes an electric field sensor, an optical microscope, and a radiometer developed to characterize the near-surface environment and study mixing by aeolian processes. MAHRS was designed to aid in the selection of optimum areas for sample collection for return to Earth.

Requirements for Portable Instrument Suites during Human Scientific Exploration of Mars.

Human explorers on the surface of Mars will have access to a far wider array of scientific tools than previous crewed planetary exploration missions, but not every tool will be compatible with the restrictions of this exploration. Spectrometers on flyby, orbital, and landed missions are currently used to determine the composition and mineralogy of geological materials of various types and sizes, from small fragments to celestial bodies in the solar system. Handheld spectrometers that are capable of in situ analyses are already used for geological exploration on Earth; however, their usefulness for human exploration missions and how data from multiple handheld instruments could be combined to enhance scientific return must be further evaluated. As part of the Biologic Analog Science Associated with Lava Terrains (BASALT) research project, we incorporated two handheld instruments, a visible-near infrared spectrometer and an X-Ray Fluorescence spectrometer, into simulated Mars exploration missions conducted on basaltic terrains in Idaho and Hawai'i. To understand the data quality provided by these handheld spectrometers, we evaluated their performance under varying conditions of measurement time, distance, angle, atmosphere, and sample matrix, and we compared data quality between handheld instruments and laboratory techniques. Here, we summarize these findings, provide guidelines and requirements on how to effectively incorporate these instruments into human exploration missions to Mars, and posit that future iterations of these instruments will be beneficial for enhancing science returned from human exploration missions.

Assessing the Acceptability of Science Operations Concepts and the Level of Mission Enhancement of Capabilities for Human Mars Exploration Extravehicular Activity.

The Biologic Analog Science Associated with Lava Terrains (BASALT) research project is investigating tools, techniques, and strategies for conducting Mars scientific exploration extravehicular activity (EVA). This has been accomplished through three science-driven terrestrial field tests (BASALT-1, BASALT-2, and BASALT-3) during which the iterative development, testing, assessment, and refinement of concepts of operations (ConOps) and capabilities were conducted. ConOps are the instantiation of operational design elements that guide the organization and flow of personnel, communication, hardware, software, and data products to enable a mission concept. Capabilities include the hardware, software, data products, and protocols that comprise and enable the ConOps. This paper describes the simulation quality and acceptability of the Mars-forward ConOps evaluated during BASALT-2. It also presents the level of mission enhancement and acceptability of the associated Mars-forward capabilities. Together, these results inform science operations for human planetary exploration.

A Catalog of Spectra, Albedos, and Colors of Solar System Bodies for Exoplanet Comparison.

We present a catalog of spectra and geometric albedos, representative of the different types of solar system bodies, from 0.45 to 2.5 µm. We analyzed published calibrated, uncalibrated spectra, and albedos for solar system objects and derived a set of reference spectra and reference albedos for 19 objects that are representative of the diversity of bodies in our solar system. We also identified previously published data that appear contaminated. Our catalog provides a baseline for comparison of exoplanet observations to 19 bodies in our own solar system, which can assist in the prioritization of exoplanets for time intensive follow-up with next-generation extremely large telescopes and space-based direct observation missions. Using high- and low-resolution spectra of these solar system objects, we also derive colors for these bodies and explore how a color-color diagram could be used to initially distinguish between rocky, icy, and gaseous exoplanets. We explore how the colors of solar system analog bodies would change when orbiting different host stars. This catalog of solar system reference spectra and albedos is available for download through the Carl Sagan Institute.

How to Detect Life on Icy Moons.

The icy moons of the outer Solar System present the possibility of subsurface water, habitable conditions, and potential abodes for life. Access to evidence that reveals the presence of life on the icy moons can be facilitated by plumes that eject material from the subsurface out into space. One instrument capable of performing life-search investigations at the icy moons is the MAss SPectrometer for Planetary EXploration/Europa (MASPEX), which constitutes a high-resolution, high-sensitivity multibounce time-of-flight mass spectrometer capable of measuring trace amounts (ppb) of organic compounds. MASPEX has been selected for the NASA Europa Clipper mission and will sample any plumes and the surface-sputtered atmosphere to assess any evidence for habitability and life. MASPEX is capable of similar investigations targeted at other icy moons. Data may be forthcoming from direct sampling but also impact dissociation because of the high speed of some analytes. Impact dissociation is analogous to the dissociation provided by modern analytical pyrolysis methods. Radiolytic dissociation on the europan surface before or during the sputtering process can also induce fragmentation similar to pyrolysis. In this study, we have compiled pyrolysis mass spectrometry data from a variety of biological and nonbiological materials to demonstrate the ability of MASPEX to recognize habitability and detect life in any plumes and atmospheres of icy moons. Key Words: Europa-Icy moons-Life detection-Mass spectrometry-Organic matter. Astrobiology 18, 843-855.