Spacecraft can carry microbial contaminants from spacecraft assembly facilities (SAFs) to the cold arid surface of Mars that may confound life detection missions or disrupt native ecosystems. Dry hygroscopic sulphate and (per)chlorate salts on Mars may absorb atmospheric humidity and deliquesce at certain times to produce dense brines, potential sources of liquid water. Microbial growth is generally prohibited under the non-permissive condition of extremely low water activity in the frigid potential brines on Mars. Here we challenged the microbial community from samples of the Jet Propulsion Laboratory SAF with the extreme chemical conditions of brines relevant to Mars. Enrichment cultures in SP medium supplemented with 50% MgSO4 or 20% NaClO3 were inoculated from washes of SAF floor wipes. Samples were taken for each of the first four weeks and then at six months after inoculation to follow changes in the SAF microbial community under high salinity for long periods. Metagenomic DNA extracts of community samples were examined by Illumina sequencing of 18S rRNA gene sequences using fungal primers. The fungal assemblage during the first month of enrichment was predominantly common Ascomycetes, primarily Saccharomyete yeasts. Basidiomycetes were detected, mainly in the Microbotryomycetes and Tremellomycetes. Fungi were much less abundant in enrichment cultures at 50% MgSO4 than at 20% NaClO3. After 6 months of enrichment, few fungi remained. Microbes persisting from the JPL SAF microbial community in aged cultures enriched at extreme salinities might be the most capable of subsequently surviving and proliferating at the near surface of Mars. The SAF fungal assemblage did not survive and proliferate as well as the SAF bacterial community.

Planetary Protection (PP) is the practice of safeguarding solar system bodies from terrestrial biological contamination and screening the Earth against potentially harmful extraterrestrial biological contamination. On Earth, cleanrooms and spacecraft surfaces are assayed using swabs and wipes that are then heat shocked for 15 min at 80°C to select for spores. The samples are further processed using the pour-plate method and Petri plates (TSA plates), with trypticase soy agar (TSA) serving as the growth medium. This sampling and processing procedure, called the NASA Standard Assay (NSA), is used by PP engineers around the world. Recent years have seen an increase in the incorporation of state-of-the-art technology, such as membrane filtration, into the NSA, with a push for implementing environmentally friendly technology into day-to-day activities. Dehydrated thin film media, such as Petrifilm Rapid Aerobic Count (RAC) plates, suit these goals as an alternative method to TSA plates. RAC plates show bacterial growth (and distinguish colonies from foreign particles such as bubbles) faster than TSA plates due to the incorporation of chromogenic colour indicators in the media. RAC plates also possess a much smaller environmental footprint than TSA plates, and are designed to evaluate even some of the challenging-to-detect environmental organisms, including spreaders that fill over 25% of the plate area in only a few hours. With these benefits in mind the PP Group at the NASA Jet Propulsion Laboratory took on the task of comparing RAC plates directly to TSA plates within the context of the NSA. Not only were the RAC plates able to detect surface environmental samples and in vitro spiked samples equivalent to NSA-processed TSA plates, but spreader organisms were countable on RAC plates at culture densities 10- to 100-fold greater than on TSA plates. In addition, RAC plates showed a robust, linear detection capability when challenged with membrane filter incorporation and organisms were easily acquired from RAC plates for archiving or post-processing experiments including MALDI-TOF bacterial identification. With their ease of use, small footprint, and both rapid and accurate bioburden measurements, RAC plates have the potential to overcome limitations posed by current PP culturing protocols.

Viruses are the most numerically abundant biological entities on Earth. As ubiquitous replicators of molecular information and agents of community change, viruses have potent effects on the life on Earth, and may play a critical role in human spaceflight, for life-detection missions to other planetary bodies and planetary protection. However, major knowledge gaps constrain our understanding of the Earth's virosphere: (1) the role viruses play in biogeochemical cycles, (2) the origin(s) of viruses and (3) the involvement of viruses in the evolution, distribution and persistence of life. As viruses are the only replicators that span all known types of nucleic acids, an expanded experimental and theoretical toolbox built for Earth's viruses will be pivotal for detecting and understanding life on Earth and beyond. Only by filling in these knowledge and technical gaps we will obtain an inclusive assessment of how to distinguish and detect life on other planetary surfaces. Meanwhile, space exploration requires life-support systems for the needs of humans, plants and their microbial inhabitants. Viral effects on microbes and plants are essential for Earth's biosphere and human health, but virus–host interactions in spaceflight are poorly understood. Viral relationships with their hosts respond to environmental changes in complex ways which are difficult to predict by extrapolating from Earth-based proxies. These relationships should be studied in space to fully understand how spaceflight will modulate viral impacts on human health and life-support systems, including microbiomes. In this review, we address key questions that must be examined to incorporate viruses into Earth system models, life-support systems and life detection. Tackling these questions will benefit our efforts to develop planetary protection protocols and further our understanding of viruses in astrobiology.

Interplanetary spacecraft are assembled with thousands of parts composed of many diverse materials. Little is known on whether any of the spacecraft materials are biocidal to the typical microbiomes that develop on spacecraft during pre-launch processing. During ongoing experiments to examine the interactive effects of solar UV irradiation, solar heating, ionizing radiation, and vacuum, we observed that bacterial spores of three Bacillus spp. were killed when incubated within small vacuum chambers for 5 days – without exposure to the aforementioned factors. Eight potential spacecraft materials were tested within the vacuum chambers for biocidal activities against spores of B. atrophaeus ATCC 9372, B. pumilus SAFR-032 and B. subtilis 168. All three species were fully inactivated (i.e., no survivors detected) by machined parts manufactured from Delrin®; a thermoplastic polyacetal polymer. Although not tested here, it is known that Delrin can off-gas formaldehyde, and thus, we hypothesize that this volatile organic compound (VOC) was responsible for the biocidal activity of the material. Knowledge of the biocidal nature of routinely used spacecraft materials might offer diverse methods to inactivate deeply embedded or shielded microbiota within spacecraft via the release of biocidal VOCs.

There is increased interest in exploring planetary bodies that have ocean worlds, and planetary protection (PP) practices for spacecraft are important to avoid harmful biological contamination of these sensitive environments. In the autumn of 2018 a diverse set of subject matter experts were assembled to examine the environmental input parameters (e.g. space radiation, Europa surface turnover) and biological input parameters that are referenced by the Europa Clipper project probabilistic risk model, to assess the < 1 × 10−4 probability of contamination requirement derived from NASA Procedural Requirements 8020.12D. A joint NASA–JPL lead workshop entitled, ‘Europa Clipper Planetary Protection Workshop’ was convened on 13–15 November 2018, to validate probability model input values, current Europa Clipper PP requirements and implementation strategy, and identify future PP research topics. The three objectives for the 3 day workshop were: (1) to validate the probability of contamination modelling framework for Europa Clipper PP; (2) to agree on probability of contamination model input values, or on a plan to derive/identify appropriate model inputs and (3) to develop workshop concurrence regarding future PP research plans and their priority. Workshop participants engaged in detailed scientific and engineering discussions focusing on Clipper mission objectives and trajectories, Europan ice shell geophysics and understanding the impact of initial microbial bioburdens and spacecraft cleanliness with all three objectives being successfully completed by the end of the workshop.

Mars exploration motivates the search for extraterrestrial life, the development of space technologies, and the design of human missions and habitations. Here, we seek new insights and pose unresolved questions relating to the natural history of Mars, habitability, robotic and human exploration, planetary protection, and the impacts on human society. Key observations and findings include:

1. – high escape rates of early Mars' atmosphere, including loss of water, impact present-day habitability;

2. – putative fossils on Mars will likely be ambiguous biomarkers for life;

3. – microbial contamination resulting from human habitation is unavoidable; and

4. – based on Mars' current planetary protection category, robotic payload(s) should characterize the local martian environment for any life-forms prior to human habitation.

Some of the outstanding questions are:

1. – which interpretation of the hemispheric dichotomy of the planet is correct;

2. – to what degree did deep-penetrating faults transport subsurface liquids to Mars' surface;

3. – in what abundance are carbonates formed by atmospheric processes;

4. – what properties of martian meteorites could be used to constrain their source locations;

5. – the origin(s) of organic macromolecules;

6. – was/is Mars inhabited;

7. – how can missions designed to uncover microbial activity in the subsurface eliminate potential false positives caused by microbial contaminants from Earth;

8. – how can we ensure that humans and microbes form a stable and benign biosphere; and

9. – should humans relate to putative extraterrestrial life from a biocentric viewpoint (preservation of all biology), or anthropocentric viewpoint of expanding habitation of space?

Studies of Mars' evolution can shed light on the habitability of extrasolar planets. In addition, Mars exploration can drive future policy developments and confirm (or put into question) the feasibility and/or extent of human habitability of space.

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.

Liquid water on Mars might be created by deliquescence of hygroscopic salts or by permafrost melts, both potentially forming saturated brines. Freezing point depression allows these heavy brines to remain liquid in the near-surface environment for extended periods, perhaps as eutectic solutions, at the lowest temperatures and highest salt concentrations where ices and precipitates do not form. Perchlorate and chlorate salts and iron sulphate form brines with low eutectic temperatures and may persist under Mars near-surface conditions, but are chemically harsh at high concentrations and were expected to be incompatible with life, while brines of common sulphate salts on Mars may be more suitable for microbial growth. Microbial growth in saturated brines also may be relevant beyond Mars, to the oceans of Ceres, Enceladus, Europa and Pluto. We have previously shown strong growth of salinotolerant bacteria in media containing 2M MgSO4 heptahydrate (~50% w/v) at 25°C. Here we extend those observations to bacterial isolates from Basque Lake, BC and Hot Lake, WA, that grow well in saturated MgSO4 medium (67%) at 25°C and in 50% MgSO4 medium at 4°C (56% would be saturated). Psychrotolerant, salinotolerant microbes isolated from Basque Lake soils included Halomonas and Marinococcus, which were identified by 16S rRNA gene sequencing and characterized phenetically. Eutectic liquid medium constituted by 43% MgSO4 at −4°C supported copious growth of these psychrotolerant Halomonas isolates, among others. Bacterial isolates also grew well at the eutectic for K chlorate (3% at −3°C). Survival and growth in eutectic solutions increases the possibility that microbes contaminating spacecraft pose a contamination risk to Mars. The cold brines of sulphate and (per)chlorate salts that may form at times on Mars through deliquescence or permafrost melt have now been demonstrated to be suitable microbial habitats, should appropriate nutrients be available and dormant cells become vegetative.

I argue that the attempts of astrobiologists and philosophers to provide an ethical justification for planetary protection policies (in particular, those aspects of policy concerning forward contamination) suffer from a ‘life bias’ in that reasons for protection are regarded as genuinely ethical only when they include some kind of direct moral consideration for extraterrestrial life. There are, I maintain, good reasons for the protection of space environments, including the protection of sites of interest to disciplines other than astrobiology. These reasons are no less ethical simply because their aim is something other than the protection of extraterrestrial life. While the possible existence of such reasons has been recognized, they have yet to be developed in a philosophically satisfying way. This paper aims to fill this lacuna by motivating and articulating an ethical perspective which recommends broader protection of the space environment. Long-range implications for such a broadening of planetary protection are considered, including implications for interstellar exploration.

The concept of sustainability is widely acknowledged as a political guideline. Economic, ecological, social and cultural aspects of sustainability are already under discussion. Current space mining efforts demand that the discussion become a broader one about ‘planetary sustainability’, including the space surrounding Earth. To date, planetary sustainability has mainly been used with reference to Earth only and I will extend it here, elaborating on a similar NASA initiative. This article (1) sketches the contemporary economic–political initiatives which call for a special reflection of Earth's location in space, and then (2) discusses the meaning of the concept of sustainability in this context. Next, (3) I relate the discussion to the issue of planetary and environmental protection, before, (4) finally, presenting a philosophical and theological perspective that seems particularly able to broach the issue of the multiple dimensions of sustainability in this context. This is the concept of constructive-critical realism. My overview of the topic concludes with (5) a summarizing outlook.

The remote operation of an asset with time-delays short enough to allow for ‘real-time’ or near real-time control – often referred to as low-latency teleoperations (LLT) – has important potential to address planetary protection concerns and to enhance astrobiology exploration. Not only can LLT assist with the search for extraterrestrial life and help mitigate planetary protection concerns as required by international treaty, but it can also aid in the real-time exploration of hazardous areas, robotically manipulate samples in real-time, and engage in precise measurements and experiments without the presence of crew in the immediate area. Furthermore, LLT can be particularly effective for studying ‘Special Regions’ – areas of astrobiological interest that might be adversely affected by forward contamination from humans or spacecraft contaminants during activities on Mars. LLT can also aid human exploration by addressing concerns about backward contamination that could impact mission details for returning Martian samples and crew back to Earth.This paper provides an overview of LLT operational considerations and findings from recent NASA analyses and workshops related to planetary protection and human missions beyond Earth orbit. The paper focuses primarily on three interrelated areas of Mars operations that are particularly relevant to the planetary protection and the search for life: Mars orbit-to-surface LLT activities; Crew-on-surface and drilling LLT; and Mars surface science laboratory LLT. The paper also discusses several additional mission implementation considerations and closes with information on key knowledge gaps identified as necessary for the advance of LLT for planetary protection and astrobiology purposes on future human missions to Mars.

The Phoenix lander at Mars polar cap found appreciable levels of (per)chlorate salts, a mixture of perchlorate and chlorate salts of Ca, Fe, Mg and Na at levels of ~0.6% in regolith. These salts are highly hygroscopic and can form saturated brines through deliquescence, likely producing aqueous solutions with very low freezing points on Mars. To support planetary protection efforts, we have measured bacterial growth tolerance to (per)chlorate salts. Existing bacterial isolates from the Great Salt Plains of Oklahoma (NaCl-rich) and Hot Lake in Washington (MgSO4-rich) were tested in high concentrations of Mg, K and Na salts of chlorate and perchlorate. Strong growth was observed with nearly all of these salinotolerant isolates at 1% (~0.1 M) (per)chlorate salts, similar to concentrations observed in bulk soils on Mars. Growth in perchlorate salts was observed at concentrations of at least 10% (~1.0 M). Greater tolerance was observed for chlorate salts, where growth was observed to 2.75 M (>25%). Tolerance to K salts was greatest, followed by Mg salts and then Na salts. Tolerances varied among isolates, even among those within the same phylogenetic clade. Tolerant bacteria included genera that also are found in spacecraft assembly facilities. Substantial microbial tolerance to (per)chlorate salts is a concern for planetary protection since tolerant microbes contaminating spacecraft would have a greater chance for survival and proliferation, despite the harsh chemical conditions found near the surface of Mars.

The differentiation between living and dead bacterial endospores is crucial in many research areas of microbiology. The identification of inactivated, non-pathogenic Bacillus anthracis spores is one reason why improvement of decontamination protocols is so desirable. Another field interested in spore viability is planetary protection, a sub-discipline of astrobiology that estimates the bioburden of spacecraft prior to launch in order to avoid interplanetary cross-contamination. We developed a dedicated, rapid and cost-effective method for identifying bacterial endospores that have been inactivated and consequently show a compromised spore wall. This novel protocol is culture-independent and is based on fluorescence microscopy and propidium monoazide (PMA) as a fluorescent marker, which is suggested to bind to DNA of spores with compromised spore coat, cortex and membranes based on our results. Inactivated preparations (treated with wet heat, irradiation, ultracentrifugation) showed a significant increase in spores that were PMA stained in their core; moreover, Bacillus atrophaeus, Bacillus safensis and Geobacillus stearothermophilus seemed to be best suited for this technique, as the spore cores of all these endospores could be positively stained after inactivation. Lastly, we describe an additional counter-staining protocol and provide an example of the application of the coupled staining methods for planetary protection purposes. The introduction of this novel protocol is expected to provide an initial insight into the various possible future applications of PMA as a non-viability marker for spores in, for example, B. anthracis-related studies, food microbiology and astrobiology.

Laboratory experiments were conducted to determine the effect of descent-engine plumes on the scouring of surface (microbial) contaminants from a spacecraft. A simulated touchdown of a half-scale lander engine and deck configuration was conducted at Mars atmospheric pressure in the NASA Ames Planetary Aeolian Laboratory. Low-density particles were used for the soil simulant to emulate the lower Martian gravity. The underside of the model had small witness plates with controlled microbial surface populations and particle impact detectors. For both steady-state engine thrust (Viking) and pulsed engine thrust (Phoenix), the exhaust plumes from the engines violently excavated the soil and produced particle-laden eddies beneath the lander that sandblasted the lander underside. The result was nearly complete erosion of microbial contaminants from the spacecraft model with their subsequent deposition in the surrounding area. It is concluded that different planetary protection cleanliness levels for different parts of a spacecraft do not necessarily prevent soil contamination because these cleaning strategies evolved without consideration of the effects of the descent engine plumes.

Atmospheric pressure glow-discharge (APGD) plasmas have been proposed for sterilizing spacecraft surfaces prior to launch. The advantages of APGD plasmas for the sterilization of spacecraft surfaces include low temperatures at treatment sites, rapid inactivation kinetics of exposed microbial cells, physical degradation and removal of microbial cells, physical removal of organic biosignature molecules, and short exposure times for the materials. However, few studies have tested APGD plasmas on spacecraft materials for their effectiveness in both sterilizing surfaces and removal of microbial cells or spores. A helium (He)+oxygen (O2) APGD plasma was used to expose six spacecraft materials (aluminum 6061, polytetrafluoroethylene (PTFE), polycarbonate, Saf-T-Vu, Rastex, and Herculite 20) doped with spores of the common spacecraft contaminant, Bacillus subtilis, for periods of time up to 6 min. Results indicated that greater than six orders of magnitude reductions in viability were observed for B. subtilis spores in as short of time as 40 s exposure to the APGD plasmas. Spacecraft materials were not affected by exposures to the APGD plasmas. However, Saf-T-Vu was the only material in which spores of B. subtilis adhered more aggressively to plasma-treated coupons when compared to non-plasma treated coupons; all other materials exhibited no significant differences between plasma and non-plasma treated coupons. In addition, spores of B. subtilis were physically degraded by exposures to the plasmas beginning at the terminal ends of spores, which appeared to be ruptured after only 30 s. After 300 s, most bacteria were removed from aluminium coupons, and only subtle residues of bacterial secretions or biofilms remained. Results support the conclusion that APGD plasmas can be used as a prelaunch cleaning and sterilization treatment on spacecraft materials provided that the biocidal and cleaning times are shorter than those required to alter surface properties of materials.

Chemical and microbiological studies of the impact of terrestrial contamination of the lunar surface during the Apollo missions could provide valuable data to help refine future Mars surface exploration plans and planetary protection requirements for a human mission to Mars. NASA and ESA have outlined new visions for solar system exploration that will include a series of lunar robotic missions to prepare for and support a human return to the Moon, and future human exploration of Mars and other destinations. Under the Committee on Space Research's (COSPAR's) current planetary protection policy for the Moon, no decontamination procedures are required for outbound lunar spacecraft. Nonetheless, future in situ investigations of a variety of locations on the Moon by highly sensitive instruments designed to search for biologically derived organic compounds would help assess the contamination of the Moon by lunar spacecraft and Apollo astronauts. These studies could also provide valuable ‘ground truth’ data for Mars sample return missions and help define planetary protection requirements for future Mars bound spacecraft carrying life detection experiments.