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.

Life Detection and Microbial Biomarker Profiling with Signs of Life Detector-Life Detector Chip During a Mars Drilling Simulation Campaign in the Hyperarid Core of the Atacama Desert.

The low organic matter content in the hyperarid core of the Atacama Desert, together with abrupt temperature shifts and high ultraviolet radiation at its surface, makes this region one of the best terrestrial analogs of Mars and one of the best scenarios for testing instrumentation devoted to in situ planetary exploration. We have operated remotely and autonomously the SOLID-LDChip (Signs of Life Detector-Life Detector Chip), an antibody microarray-based sensor instrument, as part of a rover payload during the 2019 NASA Atacama Rover Astrobiology Drilling Studies (ARADS) Mars drilling simulation campaign. A robotic arm collected drilled cuttings down to 80 cm depth and loaded SOLID to process and assay them with LDChip for searching for molecular biomarkers. A remote science team received and analyzed telemetry data and LDChip results. The data revealed the presence of microbial markers from Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Firmicutes, and Cyanobacteria to be relatively more abundant in the middle layer (40-50 cm). In addition, the detection of several proteins from nitrogen metabolism indicates a pivotal role in the system. These findings were corroborated and complemented on "returned samples" to the lab by a comprehensive analysis that included DNA sequencing, metaproteomics, and a metabolic reconstruction of the sampled area. Altogether, the results describe a relatively complex microbial community with members capable of nitrogen fixation and denitrification, sulfur oxidation and reduction, or triggering oxidative stress responses, among other traits. This remote operation demonstrated the high maturity of SOLID-LDChip as a powerful tool for remote in situ life detection for future missions in the Solar System.

A Mission Simulating the Search for Life on Mars with Automated Drilling, Sample Handling, and Life Detection Instruments Performed in the Hyperarid Core of the Atacama Desert, Chile.

We report on a field demonstration of a rover-based drilling mission to search for biomolecular evidence of life in the arid core of the Atacama Desert, Chile. The KREX2 rover carried the Honeybee Robotics 1 m depth The Regolith and Ice Drill for Exploration of New Terrains (TRIDENT) drill and a robotic arm with scoop that delivered subsurface fines to three flight prototype instruments: (1) The Signs of Life Detector (SOLID), a protein and biomolecule analyzer based on fluorescence sandwich microarray immunoassay; (2) the Planetary In Situ Capillary Electrophoresis System (PISCES), an amino acid analyzer based on subcritical water extraction coupled to microchip electrophoresis analysis; and (3) a Wet Chemistry Laboratory cell to measure soluble ions using ion selective electrodes and chronopotentiometry. A California-based science team selected and directed drilling and sampling of three sites separated by hundreds of meters that included a light-toned basin area showing evidence of aqueous activity surrounded by a rocky desert pavement. Biosignatures were detected in basin samples collected at depths ranging from 20 to 80 cm but were not detected in the surrounding area. Subsurface stratigraphy of the units drilled was interpreted from drill sensor data as fine-scale layers of sand/clay sediments interspersed with layers of harder material in the basins and a uniform subsurface composed of course-to-fine sand in the surroundings. The mission timeline and number of commands sent to accomplish each activity were tracked. The deepest sample collected (80 cm) required 55 commands, including drilling and delivery to three instruments. Elapsed time required for drilling and sample handling was less than 3 hours to collect sample from 72 cm depth, including time devoted to recovery from a jammed drill. The experiment demonstrated drilling, sample transfer technologies, and instruments that accomplished successful detection of biomolecular evidence of life in one of the most biologically sparse environments on Earth.

Towards Determining Biosignature Retention in Icy World Plumes.

With the discovery of the persistent jets of water being ejected to space from Enceladus, an understanding of the effect of the space environment on potential organisms and biosignatures in them is necessary for planning life detection missions. We experimentally determine the survivability of microbial cells in liquid medium when ejected into vacuum. Epifluorescence microscopy, using a lipid stain, and SEM imaging were used to interrogate the cellular integrity of E. coli after ejected through a pressurized nozzle into a vacuum chamber. The experimental samples showed a 94% decrease in visible intact E. coli cells but showed a fluorescence residue in the shape of the sublimated droplets that indicated the presence of lipids. The differences in the experimental conditions versus those expected on Enceladus should not change the analog value because the process a sample would undergo when ejected into space was representative. E. coli was selected for testing although other cell types could vary physiologically which would affect their response to a vacuum environment. More testing is needed to determine the dynamic range in concentration of cells expected to survive the plume environment. However, these results suggest that lipids may be directly detectable evidence of life in icy world plumes.

Simulating Mars Drilling Mission for Searching for Life: Ground-Truthing Lipids and Other Complex Microbial Biomarkers in the Iron-Sulfur Rich Río Tinto Analog.

Sulfate and iron oxide deposits in Río Tinto (Southwestern Spain) are a terrestrial analog of early martian hematite-rich regions. Understanding the distribution and drivers of microbial life in iron-rich environments can give critical clues on how to search for biosignatures on Mars. We simulated a robotic drilling mission searching for signs of life in the martian subsurface, by using a 1m-class planetary prototype drill mounted on a full-scale mockup of NASA's Phoenix and InSight lander platforms. We demonstrated fully automated and aseptic drilling on iron and sulfur rich sediments at the Río Tinto riverbanks, and sample transfer and delivery to sterile containers and analytical instruments. As a ground-truth study, samples were analyzed in the field with the life detector chip immunoassay for searching microbial markers, and then in the laboratory with X-ray diffraction to determine mineralogy, gas chromatography/mass spectrometry for lipid composition, isotope-ratio mass spectrometry for isotopic ratios, and 16S/18S rRNA genes sequencing for biodiversity. A ubiquitous presence of microbial biomarkers distributed along the 1m-depth subsurface was influenced by the local mineralogy and geochemistry. The spatial heterogeneity of abiotic variables at local scale highlights the importance of considering drill replicates in future martian drilling missions. The multi-analytical approach provided proof of concept that molecular biomarkers varying in compositional nature, preservation potential, and taxonomic specificity can be recovered from shallow drilling on iron-rich Mars analogues by using an automated life-detection lander prototype, such as the one proposed for NASA's IceBreaker mission proposal.

An Efficient Approach for Mars Sample Return Using Emerging Commercial Capabilities.

Mars Sample Return is the highest priority science mission for the next decade as recommended by the 2011 Decadal Survey of Planetary Science [1]. This article presents the results of a feasibility study for a Mars Sample Return mission that efficiently uses emerging commercial capabilities expected to be available in the near future. The motivation of our study was the recognition that emerging commercial capabilities might be used to perform Mars Sample Return with an Earth-direct architecture, and that this may offer a desirable simpler and lower cost approach. The objective of the study was to determine whether these capabilities can be used to optimize the number of mission systems and launches required to return the samples, with the goal of achieving the desired simplicity. All of the major element required for the Mars Sample Return mission are described. Mission system elements were analyzed with either direct techniques or by using parametric mass estimating relationships. The analysis shows the feasibility of a complete and closed Mars Sample Return mission design based on the following scenario: A SpaceX Falcon Heavy launch vehicle places a modified version of a SpaceX Dragon capsule, referred to as "Red Dragon", onto a Trans Mars Injection trajectory. The capsule carries all the hardware needed to return to Earth Orbit samples collected by a prior mission, such as the planned NASA Mars 2020 sample collection rover. The payload includes a fully fueled Mars Ascent Vehicle; a fueled Earth Return Vehicle, support equipment, and a mechanism to transfer samples from the sample cache system onboard the rover to the Earth Return Vehicle. The Red Dragon descends to land on the surface of Mars using Supersonic Retropropulsion. After collected samples are transferred to the Earth Return Vehicle, the single-stage Mars Ascent Vehicle launches the Earth Return Vehicle from the surface of Mars to a Mars phasing orbit. After a brief phasing period, the Earth Return Vehicle performs a Trans Earth Injection burn. Once near Earth, the Earth Return Vehicle performs Earth and lunar swing-bys and is placed into a Lunar Trailing Orbit - an Earth orbit, at lunar distance. A retrieval mission then performs a rendezvous with the Earth Return Vehicle, retrieves the sample container, and breaks the chain of contact with Mars by transferring the sample into a sterile and secure container. With the sample contained, the retrieving spacecraft makes a controlled Earth re-entry preventing any unintended release of Martian materials into the Earth's biosphere. The mission can start in any one of three Earth to Mars launch opportunities, beginning in 2022.

The sample handling system for the Mars Icebreaker Life mission: from dirt to data.

The Mars Icebreaker Life mission will search for subsurface life on Mars. It consists of three payload elements: a drill to retrieve soil samples from approximately 1 m below the surface, a robotic sample handling system to deliver the sample from the drill to the instruments, and the instruments themselves. This paper will discuss the robotic sample handling system. Collecting samples from ice-rich soils on Mars in search of life presents two challenges: protection of that icy soil--considered a "special region" with respect to planetary protection--from contamination from Earth, and delivery of the icy, sticky soil to spacecraft instruments. We present a sampling device that meets these challenges. We built a prototype system and tested it at martian pressure, drilling into ice-cemented soil, collecting cuttings, and transferring them to the inlet port of the SOLID2 life-detection instrument. The tests successfully demonstrated that the Icebreaker drill, sample handling system, and life-detection instrument can collectively operate in these conditions and produce science data that can be delivered via telemetry--from dirt to data. Our results also demonstrate the feasibility of using an air gap to prevent forward contamination. We define a set of six analog soils for testing over a range of soil cohesion, from loose sand to basalt soil, with angles of repose of 27° and 39°, respectively. Particle size is a key determinant of jamming of mechanical parts by soil particles. Jamming occurs when the clearance between moving parts is equal in size to the most common particle size or equal to three of these particles together. Three particles acting together tend to form bridges and lead to clogging. Our experiments show that rotary-hammer action of the Icebreaker drill influences the particle size, typically reducing particle size by ≈ 100 µm.

The Icebreaker Life Mission to Mars: a search for biomolecular evidence for life.

The search for evidence of life on Mars is the primary motivation for the exploration of that planet. The results from previous missions, and the Phoenix mission in particular, indicate that the ice-cemented ground in the north polar plains is likely to be the most recently habitable place that is currently known on Mars. The near-surface ice likely provided adequate water activity during periods of high obliquity, ≈ 5 Myr ago. Carbon dioxide and nitrogen are present in the atmosphere, and nitrates may be present in the soil. Perchlorate in the soil together with iron in basaltic rock provides a possible energy source for life. Furthermore, the presence of organics must once again be considered, as the results of the Viking GCMS are now suspect given the discovery of the thermally reactive perchlorate. Ground ice may provide a way to preserve organic molecules for extended periods of time, especially organic biomarkers. The Mars Icebreaker Life mission focuses on the following science goals: (1) Search for specific biomolecules that would be conclusive evidence of life. (2) Perform a general search for organic molecules in the ground ice. (3) Determine the processes of ground ice formation and the role of liquid water. (4) Understand the mechanical properties of the martian polar ice-cemented soil. (5) Assess the recent habitability of the environment with respect to required elements to support life, energy sources, and possible toxic elements. (6) Compare the elemental composition of the northern plains with midlatitude sites. The Icebreaker Life payload has been designed around the Phoenix spacecraft and is targeted to a site near the Phoenix landing site. However, the Icebreaker payload could be supported on other Mars landing systems. Preliminary studies of the SpaceX Dragon lander show that it could support the Icebreaker payload for a landing either at the Phoenix site or at midlatitudes. Duplicate samples could be cached as a target for possible return by a Mars Sample Return mission. If the samples were shown to contain organic biomarkers, interest in returning them to Earth would be high.

Science results from a Mars drilling simulation (Río Tinto, Spain) and ground truth for remote science observations.

Science results from a field-simulated lander payload and post-mission laboratory investigations provided "ground truth" to interpret remote science observations made as part of the 2005 Mars Astrobiology Research and Technology Experiment (MARTE) drilling mission simulation. The experiment was successful in detecting evidence for life, habitability, and preservation potential of organics in a relevant astrobiological analogue of Mars. SCIENCE RESULTS: Borehole 7 was drilled near the Río Tinto headwaters at Peña de Hierro (Spain) in the upper oxidized remnant of an acid rock drainage system. Analysis of 29 cores (215 cm of core was recovered from 606 cm penetrated depth) revealed a matrix of goethite- (42-94%) and hematite-rich (47-87%) rocks with pockets of phyllosilicates (47-74%) and fine- to coarse-grained loose material. Post-mission X-ray diffraction (XRD) analysis confirmed the range of hematite:goethite mixtures that were visually recognizable (approximately 1:1, approximately 1:2, and approximately 1:3 mixtures displayed a yellowish-red color whereas 3:1 mixtures displayed a dark reddish-brown color). Organic carbon was poorly preserved in hematite/goethite-rich materials (C(org) <0.05 wt %) beneath the biologically active organic-rich soil horizon (C(org) approximately 3-11 wt %) in contrast to the phyllosilicate-rich zones (C(org) approximately 0.23 wt %). GROUND TRUTH VS. REMOTE SCIENCE ANALYSIS: Laboratory-based analytical results were compared to the analyses obtained by a Remote Science Team (RST) using a blind protocol. Ferric iron phases, lithostratigraphy, and inferred geologic history were correctly identified by the RST with the exception of phyllosilicate-rich materials that were misinterpreted as weathered igneous rock. Adenosine 5'-triphosphate (ATP) luminometry, a tool available to the RST, revealed ATP amounts above background noise, i.e., 278-876 Relative Luminosity Units (RLUs) in only 6 cores, whereas organic carbon was detected in all cores. Our manned vs. remote observations based on automated acquisitions during the project provide insights for the preparation of future astrobiology-driven Mars missions.

SOLID2: an antibody array-based life-detector instrument in a Mars Drilling Simulation Experiment (MARTE).

A field prototype of an antibody array-based life-detector instrument, Signs Of LIfe Detector (SOLID2), has been tested in a Mars drilling mission simulation called MARTE (Mars Astrobiology Research and Technology Experiment). As one of the analytical instruments on the MARTE robotic drilling rig, SOLID2 performed automatic sample processing and analysis of ground core samples (0.5 g) with protein microarrays that contained 157 different antibodies. Core samples from different depths (down to 5.5 m) were analyzed, and positive reactions were obtained in antibodies raised against the Gram-negative bacterium Leptospirillum ferrooxidans, a species of the genus Acidithiobacillus (both common microorganisms in the Río Tinto area), and extracts from biofilms and other natural samples from the Río Tinto area. These positive reactions were absent when the samples were previously subjected to a high-temperature treatment, which indicates the biological origin and structural dependency of the antibody-antigen reactions. We conclude that an antibody array-based life-detector instrument like SOLID2 can detect complex biological material, and it should be considered as a potential analytical instrument for future planetary missions that search for life.

The subsurface geology of Río Tinto: material examined during a simulated Mars drilling mission for the Mars Astrobiology Research and Technology Experiment (MARTE).

The 2005 Mars Astrobiology Research and Technology Experiment (MARTE) project conducted a simulated 1-month Mars drilling mission in the Río Tinto district, Spain. Dry robotic drilling, core sampling, and biological and geological analytical technologies were collectively tested for the first time for potential use on Mars. Drilling and subsurface sampling and analytical technologies are being explored for Mars because the subsurface is the most likely place to find life on Mars. The objectives of this work are to describe drilling, sampling, and analytical procedures; present the geological analysis of core and borehole material; and examine lessons learned from the drilling simulation. Drilling occurred at an undisclosed location, causing the science team to rely only on mission data for geological and biological interpretations. Core and borehole imaging was used for micromorphological analysis of rock, targeting rock for biological analysis, and making decisions regarding the next day's drilling operations. Drilling reached 606 cm depth into poorly consolidated gossan that allowed only 35% of core recovery and contributed to borehole wall failure during drilling. Core material containing any indication of biology was sampled and analyzed in more detail for its confirmation. Despite the poorly consolidated nature of the subsurface gossan, dry drilling was able to retrieve useful core material for geological and biological analysis. Lessons learned from this drilling simulation can guide the development of dry drilling and subsurface geological and biological analytical technologies for future Mars drilling missions.

Underground habitats in the Río Tinto basin: a model for subsurface life habitats on Mars.

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

Visible-near infrared point spectrometry of drill core samples from Río Tinto, Spain: results from the 2005 Mars Astrobiology Research and Technology Experiment (MARTE) drilling exercise.

Sampling of subsurface rock may be required to detect evidence of past biological activity on Mars. The Mars Astrobiology Research and Technology Experiment (MARTE) utilized the Río Tinto region, Spain, as a Mars analog site to test dry drilling technologies specific to Mars that retrieve subsurface rock for biological analysis. This work examines the usefulness of visible-near infrared (VNIR) (450-1000 nm) point spectrometry to characterize ferric iron minerals in core material retrieved during a simulated Mars drilling mission. VNIR spectrometry can indicate the presence of aqueously precipitated ferric iron minerals and, thus, determine whether biological analysis of retrieved rock is warranted. Core spectra obtained during the mission with T1 (893-897 nm) and T2 (644-652 nm) features indicate goethite-dominated samples, while relatively lower wavelength T1 (832-880 nm) features indicate hematite. Hematite/goethite molar ratios varied from 0 to 1.4, and within the 880-898 nm range, T1 features were used to estimate hematite/goethite molar ratios. Post-mission X-ray analysis detected phyllosilicates, which indicates that examining beyond the VNIR (e.g., shortwave infrared, 1000-2500 nm) will enhance the detection of other minerals formed by aqueous processes. Despite the limited spectral range of VNIR point spectrometry utilized in the MARTE Mars drilling simulation project, ferric iron minerals could be identified in retrieved core material, and their distribution served to direct core subsampling for biological analysis.

The 2005 MARTE Robotic Drilling Experiment in Río Tinto, Spain: objectives, approach, and results of a simulated mission to search for life in the Martian subsurface.

The Mars Astrobiology Research and Technology Experiment (MARTE) simulated a robotic drilling mission to search for subsurface life on Mars. The drill site was on Peña de Hierro near the headwaters of the Río Tinto river (southwest Spain), on a deposit that includes massive sulfides and their gossanized remains that resemble some iron and sulfur minerals found on Mars. The mission used a fluidless, 10-axis, autonomous coring drill mounted on a simulated lander. Cores were faced; then instruments collected color wide-angle context images, color microscopic images, visible-near infrared point spectra, and (lower resolution) visible-near infrared hyperspectral images. Cores were then stored for further processing or ejected. A borehole inspection system collected panoramic imaging and Raman spectra of borehole walls. Life detection was performed on full cores with an adenosine triphosphate luciferin-luciferase bioluminescence assay and on crushed core sections with SOLID2, an antibody array-based instrument. Two remotely located science teams analyzed the remote sensing data and chose subsample locations. In 30 days of operation, the drill penetrated to 6 m and collected 21 cores. Biosignatures were detected in 12 of 15 samples analyzed by SOLID2. Science teams correctly interpreted the nature of the deposits drilled as compared to the ground truth. This experiment shows that drilling to search for subsurface life on Mars is technically feasible and scientifically rewarding.