The National Aeronautics and Space Administration (NASA) and the European Space Agency (ESA) are studying how samples might be brought back to Earth from Mars safely. Backward planetary protection is key in this complex endeavour, as it is required to prevent potential adverse effects from returning materials to Earth's biosphere. As the question of whether or not life exists on Mars today or whether it ever did in the past is still unanswered, the effort to return samples from Mars is expected to be categorized as a ‘Restricted Earth Return’ mission, for which NASA policy requires the containment of any unsterilized material returned to Earth. NASA is investigating several solutions to contain Mars samples and sterilize any uncontained Martian particles. This effort has significant implications for both NASA's scientific mission, and the Earth's environment; and so special care and vigilance are needed in planning and execution in order to assure acceptance of safety to Earth's biosphere. To generate a technically acceptable sterilization process across a wide array of scientific and other stakeholders, on 30–31 January 2019, 10–11 June 2019 and 19–20 February 2020, NASA informally convened a Sterilization Working Group (SWG) composed of experts from industry, academia and government to assess methods for sterilization and inactivation, to identify future work needed to verify these methods against biological challenges, and to determine their feasibility for implementation on robotic spacecraft in deep space. The goals of the SWG were:

1. (1) Understand what it means to sterilize and/or inactivate Martian materials and how that understanding can be applied to the Mars Sample Return (MSR) mission.

2. (2) Assess methods for sterilization and inactivation, and identify future work needed to verify these methods.

3. (3) Provide an effective plan for communicating with other agencies and the public.

This paper provides a summary of the discussions and conclusions of the SWG over these three workshops. It reflects a consensus position based on qualitative discussion of how agencies might approach the problem of sterilization of Mars material. The SWG reached a consensus that sterilization options can be considered on the basis of biology as we know it, and that sterilization modalities that are effective on terrestrial materials and organisms should be part of the MSR planetary protection strategy. Conclusions pointed to several industry standards for sterilization to include heat, chemical, UV radiation and low-heat plasma. Technical trade-offs for each sterilization modality were discussed while simultaneously considering the engineering challenges and limitations for spaceflight. Future work includes more in-depth discussions on technical trade-offs of sterilization modalities, identifying and testing Earth analogue challenge organisms and proteinaceous molecules against chosen modalities, and executing collaborative agreements between NASA and external working group partners to help close data gaps, and to establish strong, scientifically grounded sterilization and inactivation standards for MSR.

Previously conducted space missions revealed the presence of perchlorates, which are known to have a high oxidizing potential in Martian regolith, at the level of 0.5%. Due to hygroscopic properties and crystallization features of perchlorate-containing solutions, assumptions leading to the possibility of the existence of liquid water in the form of brines, which can contribute to the vital activity of microorganisms, have been made. At the same time, high concentrations of perchlorates can inhibit the growth of microorganisms and cause their death. Previously performed studies have discovered the presence of highly diverse microbial communities in terrestrial perchlorate-containing soils and have also demonstrated the stability and activity of some prokaryotes cultured on highly concentrated perchlorates media (over 10%). Nevertheless, the limits of microbial tolerance to perchlorates and whether microbial communities are able to withstand the effects of high concentrations of perchlorates remain uncertain. The aim of this research was to study the reaction of microbial communities of hot-arid and cryo-arid soils and sedimentary rocks to the adding of a highly concentrated solution of sodium perchlorate (5%) in situ. An increase in the total number of prokaryotes, the number of metabolically active Bacteria and Archaea, and the variety of the consumed substrates were revealed. It was observed that in samples incubated with sodium perchlorate, a high taxonomic diversity of the microbial community is preserved at a level comparable to control sample. The study shows that the presence of high concentrations of sodium perchlorate (5%) in the soil does not lead to the death or significant inhibition of microbial communities.

The presence of perchlorate in the Martian soil may limit in-situ resource utilization (ISRU) technologies to support human outposts. In order to exploit the desiccation, radiation-tolerant cyanobacterium Chroococcidopsis in Biological Life Support Systems based on ISRU, we investigated the perchlorate tolerance of Chroococcidopsis sp. CCMEE 029 and its derivative CCMEE 029 P-MRS. This strain was obtained from dried cells mixed with Martian regolith simulant and exposed to Mars-like conditions during the BIOMEX space experiment. After a 55-day exposure of up to 200 mM perchlorate ions, a tolerance threshold value of 100 mM perchlorate ions was identified for both Chroococcidopsis strains. After 40-day incubation, a Mars-relevant perchlorate concentration of 2.4 mM perchlorate ions, provided as a 60 and 40% mixture of Mg- and Ca-perchlorate, had no negative effect on the growth rate of the two strains. A proof-of-concept experiment was conducted using Chroococcidopsis lysate in ISRU technologies to feed a heterotrophic bacterium, i.e. an Escherichia coli strain capable of metabolizing sucrose. The sucrose content was fivefold increased in Chroococcidopsis cells through air-drying and the yielded lysate successfully supported the bacterial growth. This suggested that Chroococcidopsis is a suitable candidate for ISRU technologies to support heterotrophic BLSS components in a Mars-relevant perchlorate environment that would prove challenging to many other cyanobacteria, allowing a ‘live off the land’ approach on Mars.

Zagami is a basaltic shergottite well characterized among Martian meteorites. For this reason, it is used as a reference sample to verify the feasibility of the vibrational spectroscopy techniques. In general, these techniques seeking extract spectral signatures from individual mineral present in the meteorite samples. Among some FTIR results were detected organic compounds in the Zagami meteorite and recorded in some articles. Based on these results, an analysis of a Zagami meteorite sample was performed using FTIR and Raman spectrometry. Examining the fragment of Zagami meteorite using the FTIR technique, it was possible to identify the same band of 2920 and 2850 cm−1 described by other references, which were interpreted as organic components and were recorded such as aliphatic hydrocarbon. Another unusual result in Martian meteorite was the detection of the 1300 cm−1 band using Raman. The main reason for this peculiarity is that band is characterized in meteorites as a D band. Generally, this band is attributed to structural defects and the disordering of carbon. However, care should be taken when interpreting Raman spectra around 1300 cm−1 and FTIR spectra 2950–2840 cm−1 of the sample from oxidizing environments such as Mars. Misconceptions can occur during the interpretations of the bands, and this can lead to incorrect identification. Thus, an analysis of the mentioned peaks, such as chemical and mineralogical assignments, will be provided and suggested in order to compare the results that mentioned the presence of organic compounds in the Zagami meteorite.

The Kepler data show that habitable small planets orbiting Red Dwarf stars (RDs) are abundant, and hence might be promising targets to look at for biomarkers and life. Planets orbiting within the habitable zone of RDs are close enough to be tidally locked. Some recent works have cast doubt on the ability of planets orbiting RDs to support life. In contrast, it is shown that temperatures suitable for liquid water and even for organic molecules may exist on tidally locked planets (TLPs) of RDs for a wide range of atmospheres. We chart the surface temperature distribution as a function of the irradiation, greenhouse factor and heat circulation. The habitability boundaries and their dependence on the atmospheric properties are derived. By extending our previous analyses of TLPs, we find that tidally locked as well as synchronous (not completely locked) planets of RDs and K-type stars may support life, for a wider range of orbital distance and atmospheric conditions than previously thought. In particular, it is argued that life clement environments may be possible on tidally locked and synchronously orbiting planets of RDs and K-type stars, with conditions supporting oxygenic photosynthesis, which on Earth was a key to complex life. Different climate projections and the biological significance of tidal locking on putative complex life are reviewed. We show that when the effect of continuous radiation is taken into account, the photo-synthetically active radiation available on TLPs, even of RDs, could produce a high-potential plant productivity, in analogy to mid-summer growth at high latitudes on Earth. Awaiting the findings of TESS and JWST, we discuss the implications of the above arguments to the detection of biomarkers such as liquid water and oxygen, as well as to the abundance of biotic planets and life.

This paper describes strategies to search for, detect, and identify organic material on the surface and subsurface of Mars. The strategies described include those applied by landed missions in the past and those that will be applied in the future. The value and role of ESA's ExoMars rover and of her key science instrument Mars Organic Molecule Analyzer (MOMA) are critically assessed.

Mars landed and orbiter missions have instrumentation capable of detecting oxychlorine phases (e.g. perchlorate, chlorate) on the surface. Perchlorate (~0.6 wt%) was first detected by the Wet Chemistry Laboratory in the surface material at the Phoenix Mars Landing site. Subsequent analyses by the Thermal Evolved Gas Analyser aboard the same lander detected an oxygen release (~465°C) consistent with the thermal decomposition of perchlorate. Recent thermal analysis by the Mars Science Laboratory's Sample Analysis at Mars instrument has also indicated the presence of oxychlorine phases (up to 1.2 wt%) in Gale Crater materials. Despite being at detectable concentrations, the Chemistry and Mineralogy (CheMin) X-ray diffractometer has not detected oxychlorine phases. This suggests that Gale Crater oxychlorine may exist as poorly crystalline phases or that perchlorate/chlorate mixtures exist, so that individual oxychlorine concentrations are below CheMin detection limits (~1 wt%). Although not initially designed to detect oxychlorine phases, reinterpretation of Viking Gas Chromatography/Mass Spectrometer data also suggest that oxychlorine phases are present in the Viking surface materials. Remote near-infrared spectral analyses by the Compact Reconnaissance Imaging Spectrometer for Mars (CRISM) instrument indicate that at least some martian recurring slope lineae (RSL) have spectral signatures consistent with the presence of hydrated perchlorates or chlorates during the seasons when RSL are most extensive. Despite the thermal emission spectrometer, Thermal Emission Imaging System, Observatoire pour la Minéralogie, l'Eau, les Glaces et l'Activité and CRISM detection of hundreds of anhydrous chloride (~10–25 vol%) deposits, expected associated oxychlorine phases (>5–10 vol%) have not been detected. Total Cl and oxychlorine data sets from the Phoenix Lander and the Mars Science Laboratory missions could be used to develop oxychlorine versus total Cl correlations, which may constrain oxychlorine concentrations at other locations on Mars by using total Cl determined by other missions (e.g. Viking, Pathfinder, MER and Odyssey). Development of microfluidic or ‘lab-on-a-chip’ instrumentation has the potential to be the next generation analytical capability used to identify and quantify individual oxychlorine species on future landed robotic missions to Mars.

Various Mars missions have detected Cl atoms, chlorides and perchlorates in martian surface materials. The global soils, in particular, always contain significant levels of observable Cl. Direct evidence points to this Cl being in the form of both chlorides and perchlorates, and possibly also chlorates and other oxychlorines. The most widespread measurements have been of Cl atoms, and cannot discern the chemical form. However, from separate evidence of perchlorate obtained at high latitudes (Phoenix lander) and low latitudes (Curiosity rover), it is likely that perchlorates are widespread, albeit in varying proportions relative to the total amount of ubiquitous Cl.

The permanently frozen volcanic sediment is one of the most promising geological objects for searching life on Mars. On Earth, volcanic intrusions into permafrost result in formation of the unique microbial communities. We propose several terrestrial analogues of Martian polar volcanoes, such as the permanently frozen volcanic sediments on the Kamchatka peninsula and in Antarctica. The present study shows applicability of the morphometric analysis for demonstration of the morphological similarity between the terrestrial and Martian cinder cones. In the present work, the morphometric analysis of young Martian landforms is based on the assumption that the conical structures identified on digital terrain model (DTM) are volcanic cinder cones. Morphometric analysis of the studied cones showed a range of degradation. The extent of degradation may be an indicator of age based on comparison with volcanic cinder cones on Earth. A morphometric analysis of potentially young volcanic cones in the North Polar Region of Mars was performed to estimate their relative age. The 14 potential cinder cones were identified using the DTM provided by Mars Express High Resolution Stereo Camera (HRSC), allowing for the basic morphometric calculations. The majority of the cinder cones are localized in the Chasma Boreale region within the area 79°–81°N and 261°–295°E. The calculated morphometric parameters showed that the cone average steepness varied from 3.4° to 11.8°, cone height-to-width ratio varied from 0.025 to 0.12, and the ratio between surface and basal area of the cone varied from 1.005 to 1.131. The studied cinder cones were classified with respect to the morphometric ratios assuming that larger values correspond to the younger structures. Employing the terrestrial analogy of morphometric ratios as a proxy for relative geological age, we suggest that existing microorganisms may be found in permafrost of young Martian cinder cones.

Perchlorate (ClO4−) is widespread in Martian soils at concentrations between 0.5 and 1%. At such concentrations, perchlorate could be an important source of oxygen, but it could also become a critical chemical hazard to astronauts. In this paper, we review the dual implications of ClO4− on Mars, and propose a biochemical approach for removal of perchlorate from Martian soil that would be energetically cheap, environmentally friendly and could be used to obtain oxygen both for human consumption and to fuel surface operations.

If confirmed, the extremely low concentrations of methane (CH4) detected in the Martian atmosphere may represent reservoirs and emission processes that would normally be considered negligible on Earth. One such process is the release of ancient volatiles from fluid inclusions and interstitial sites in rocks and minerals during erosion or geothermal activity. Using a highly sensitive rock-crushing and mass-spectrometry technique previously shown to detect CH4 in serpentinites and hydrothermal mineral deposits, we have demonstrated that CH4 and other ancient volatiles can be recovered from basalt, the dominant rock type on the Martian surface. Basalt samples from a wide range of ages and geological systems were tested, all of which released CH4 when crushed. Oxidative weathering was associated with lower quantities of CH4. Otherwise, CH4 recoverability showed no relationship with age or geological context. Mineral veins, cross-cutting one locality were found to share the volatile composition of the basalt. In general, the results suggest that CH4-release from ancient basalts could be a significant process on Mars, which could be further investigated by Martian rovers using a similar rock-crushing and mass spectrometry technique in situ.

At present, bulk liquid water on the surface and near-subsurface of Mars does not exist due to the scarcity of condensed- and gas-phase water, pressure and temperature constraints. Given that the nuclei of soil and ice, that is, the soil solid and ice lattice, respectively, are coated with adsorbed and/or thin liquid films of water well below 273 K and the availability of water limits biological activity, we quantify lower and upper limits for the thickness of such adsorbed/water films on the surface of the Martian regolith and for subsurface ice. These limits were calculated based on experimental and theoretical data for pure water ice and water ice containing impurities, where water ice containing impurities exhibit thin liquid film enhancements, ranging from 3 to 90. Close to the cold limit of water stability (i.e. 273 K), thin liquid film thicknesses at the surface of the Martian regolith is 0.06 nm (pure water ice) and ranges from 0.2 to 5 nm (water ice with impurities). An adsorbed water layer of 0.06 nm implies a dessicated surface as the thickness of one monolayer of water is 0.3 nm but represents 0.001–0.02% of the Martian atmospheric water vapour inventory. Taking into account the specific surface area (SSA) of surface-soil (i.e. top 1 mm of regolith and 0.06 nm adsorbed water layer), shows Martian surface-soil may contain interfacial water that represents 6–66% of the upper- and lower-limit atmospheric water vapour inventory and almost four times and 33%, the lower- and upper-limit Martian atmospheric water vapour inventory. Similarly, taking the SSA of Martian soil, the top 1 mm or regolith at 5 nm thin liquid water thickness, yields 1.10×1013 and 6.50×1013 litres of waters, respectively, 55–325 times larger than Mars’ atmospheric water vapour inventory. Film thicknesses of 0.2 and 5 nm represent 2.3×104–1.5×106 litres of water, which is 6.0×10−7–4.0×10−4%, respectively, of a 10 pr μm water vapour column, and 3.0×10−6–4.0×10−4% and 6.0×10−6–8.0×10−4%, respectively, of the Martian atmospheric water vapour inventory. Thin liquid film thicknesses on/in subsurface ice were investigated via two scenarios: (i) under the idealistic case where it is assumed that the diurnal thermal wave is equal to the temperature of ice tens of centimetres below the surface, allowing for such ice to experience temperatures close to 273 K and (ii) under the, likely, realistic scenario where the diurnal thermal wave allows for the maximum subsurface ice temperature of 235 K at 1 m depth between 30°N and 30°S. Scenario 1 yields thin liquid film thicknesses ranging from 11 to 90 nm; these amounts represent 4×106–3.0×107 litres of water. For pure water ice, Scenario 2 reveals that the thickness of thin liquid films contained on/within Martian subsurface is less than 1.2 nm, several molecular layers thick. Conversely, via the effect of impurities at 235 K allows for a thin liquid film thickness on/within subsurface ice of 0.5 nm, corresponding to 6.0×104 litres of water. The existence of thin films on Mars is supported by data from the Mars Exploration Rovers (MERs) Spirit and Opportunity's Alpha Proton X-ray Spectrometer instrumentation, which have detected increased levels of bromine beneath the immediate surface, suggestive of the mobilization of soluble salts by thin films of liquid water towards local cold traps. These findings show that biological activity on the Martian surface and subsurface is not limited by nanometre dimensions of available water.