What are the potential problems with bringing Martian samples to Earth?

NASA and other space-faring agencies and nations are levying requirements for the safe return of samples from extraterrestrial bodies as per the obligations outlined in the Outer Space Treaty and are expending significant effort to mitigate any potential risks to Earth's biosphere from the return of samples from extraterrestrial bodies. Although adverse consequences could conceivably result from the introduction of extraterrestrial material into Earth's biosphere (as will be discussed below), the SWG concluded, as similar working groups sponsored by the National Research Council (NRC) and the European Science Foundation (ESF) have previously done concerning returning samples from Mars (National Research Council 2009 and 1997; Ammann et al. Reference Ammann, Baross, Bennett, Bridges, Fragola, Kerrest, Marshall-Bowman, Raoul, Rettberg, Rummel, Salminen, Stackebrandt and Walter2012), the likelihood of such a risk transpiring is considered to be extremely low. Specifically, these past reports have noted that:

While it is impossible to remove all risk without ceasing space exploration, further analysis of BTC containment and inactivation strategies is essential to provide crucial data on the claim that a MSR mission could achieve a very high probability of not releasing unsterilized particles. There is always some level of risk associated with exploration into the unknown, and it was the goal of the SWG to help manage the risks of possible adverse effects to the Earth's biosphere while maintaining the science integrity of the returned samples.

The Martian environment and the potential for life

Seeking evidence of life from extraterrestrial sources is one of the great scientific goals and challenges of our time. One of the best ways we can address this is by exploring Mars, an object that shared with Earth a similar early geological history, particularly during the time when life appeared on our planet. If life ever arose on the Red Planet, it probably did when Mars was wetter, sometime within the first half billion years after planetary formation (Nisbet and Sleep Reference Nisbet and Sleep2001; Zahnle et al. Reference Zahnle, Arndt, Cockell, Halliday, Nisbet, Selsis and Sleep2006). Conditions then were similar to those when life gained a foothold on the young Earth prior to 3.8 billion years ago. This makes Mars a primary target to search for signs of life in our Solar System.

It is likely that 4.45 billion years ago, early Mars also had developed a global ocean (or large bodies of water) enveloped in a 1 bar, mostly CO₂ atmosphere (Elkins-Tanton Reference Elkins-Tanton2011). However, Mars is much further away from the Sun than Earth, and is smaller; therefore, in the absence of other inputs, Mars would have quickly frozen over (Fairén et al. Reference Fairén, Haqq-Misra and McKay2012). But, as on Earth, we can expect that active sub-surface hydrothermal processes driven by internal heat may have helped raise surface temperatures by releasing CO₂, CH₄ and other gases (Pavlov et al. Reference Pavlov, Kasting, Brown, Rages and Freedman2000; Oze and Sharma Reference Oze and Sharma2005; Schulte et al. Reference Schulte, Richards, Daly, Kurz, McDonald and Holden2006). The extensive subglacial, submerged and emerged volcanic/hydrothermal activity would have resulted in numerous liquid water-rich settings (Warner and Farmer (Reference Warner and Farmer2010); Cousins and Crawford, Reference Cousins and Crawford2011). The right mixture of ingredients, temperature and chemical gradients, organic molecule transport and concentration, and fixation processes could have been found just as well in a plethora of terrestrial submarine vents or in a multitude of vents under top-frozen Martian bodies of water (Westall et al. Reference Westall, Loizeau, Foucher, Bost, Betrand, Vago and Kminek2013; Russell et al. Reference Russell, Barge, Bhartia, Bocanegra, Bracher, Branscomb, Kidd, McGlynn, Meier, Nitschke, Shibuya, Vance, White and Kanik2014).

Although extraterrestrial life could conceivably be different from life on Earth, the SWG concurred that it is most likely to be carbon and water based, or at the very least to share Earth's fundamental chemistries (e.g. covalent and ionic bonds) (Berg et al. Reference Berg, Tymoczko, Gatto and Stryer2019). Additional information on the use of fundamental chemistries is included subsequently in this paper.

Life originated on Earth prior to 3.8 billion years ago; and if life originated on Mars, it probably arose during the same time-period given the similarities in the genesis of the two planets. With regards to life on Mars, there are several scenarios that could have occurred, including:

• • Mars has always been a lifeless planet.

• • Life originated on Mars independent of life on Earth and went extinct or is extant in diverse refugia in the lithosphere.

• • Life originated on Mars and, during interplanetary traffic of rocky material in the early solar system (Bottke and Norman Reference Bottke and Norman2017), and subsequently was transported to Earth.

• • Life originated on Earth and, during interplanetary traffic of rocky material in the early solar system, and was transported to Mars.

If life originated on Mars and was not transported to Earth it might bear little resemblance to life on Earth, but it still most likely shares our fundamental chemistries of chemical bonds. The detection of divergent life on Mars would reveal a true second genesis of life in the universe. Thus, any life detected on Mars might not only prove that there is life on other planets but it might also influence our models for the origin of life on Earth. If there is life on Mars, there are three scenarios under which life on Mars and Earth may both use DNA and RNA (or analogous nucleic acids as hereditary materials):

1. (1) Transfer of life between the two planets.

2. (2) Transfer of life from a more distant common source to both planets.

3. (3) Independent development of the same DNA/RNA-based life forms.

Because life does exist on Earth, transfer between the planets would be the most reasonable of these scenarios if indeed life ever existed on Mars. Common ancestry of life on Earth and Mars would be dependent upon impact events (e.g. meteorites, comets, asteroids) resulting in ejection, transit through high radiation interplanetary space, entry and adaptation to another world. Recent studies suggest Martian meteorites were transferred to the Earth at shortened time scales and with higher fluxes than previously believed (Gladman and Burns Reference Gladman and Burns1996; Gladman et al. Reference Gladman, Migliorini, Morbidelli, Zappalà, Michel, Cellino, Froeschlé, Levison, Bailey and Duncan1997; Mileikowsky et al. Reference Mileikowsky, Cucinotta, Wilson, Gladman, Horneck, Lindegren, Melosh, Rickman, Valtonen and Zhengi2000). The final destination of 7% of Martian meteorites is thought to be the Earth, delivering an estimated one billion tons of debris over the history of the solar system (Gladman and Burns Reference Gladman and Burns1996). Several dozen SNC meteorites (i.e. meteorites named after the locations where examples of these meteorites were first found – Shergotty (India), Nakhla (Egypt) and Chassigny (France)) of Martian origin have been discovered here on Earth, and analyses indicate that 20% of Martian meteorites have only experienced mild internal heating during ejection and impact (Weiss et al. Reference Weiss, Kirschvink, Baudenbacher, Vali, Peters, Macdonald and Wikswo2000; Fritz et al. Reference Fritz, Artemieva and Greshake2005; Shuster and Weiss Reference Shuster and Weiss2005). Low internal heating during ejection occurs because of interference between the impact shock wave and the reflected shock wave, leading to lower shock pressures but higher velocities near the target surface. Empirical studies showed that certain types of microbes could survive the requisite shock pressures (Gratz et al. Reference Gratz, Nellis and Hinsey1993; Horneck et al. Reference Horneck, Stöffler, Ott, Hornemann, Cockell, Moeller, Meyer, Vera, Fritz, Schade and Artemieva2008). Atmospheric entry heats the surfaces to unsurvivable temperatures, but because atmospheric transit occurs so quickly, the internal temperatures of meteorites can stay quite moderate below 100°C consistent with the observations of ALH84001 (Fritz et al. Reference Fritz, Artemieva and Greshake2005) where intact amino acids were found. Once life had evolved on one planet, this meteoritic transfer rate makes it plausible that the adjacent planets could ‘catch’ life rather than independently evolve life (Davies Reference Davies2003).

Of the more than 60 000 meteorites that have been catalogued on Earth, 246 have been identified as originating from Mars, including one weighting 18 kg that fell in 1962 (Gladman and Burns Reference Gladman and Burns1996). Thus, the few hundred grams of Martian dust that would be delivered by the MSR would not be the first Martian material to be transferred to Earth. As discussed above, there has been significant, previous meteoritic transfer of Martian materials to Earth, and Earth materials to Mars. Therefore, it is plausible that Earth has been previously exposed to Martian microbiota (if present) and that the Martian material has already had the opportunity to ‘contaminate’ Earth's biosphere yet has caused no known effects.

Two major challenges during transit are desiccation and radiation. Desiccation tolerance may be enhanced through entombment in salt crystals or rock fissures. Certain types of dormant Earth-based microorganisms can show dramatic resistance to drying and heat inactivation when encased on crystals (Doyle and Ernst, Reference Doyle and Ernst1967). UV is easily shielded by tens of microns of material, as evidenced by low survival of single layers of microorganisms but high survival of thicker biofilms of Bacillus subtilis (including dormant spores) exposed to space for 1.5 years on-board the International Space Station (Horneck et al. Reference Horneck, Moeller, Thierry, Mancinelli, Nicholson, Panitz, Rabbow, Rettberg, Spry, Stackebrandt, Vaishampayan and Venkateswaran2012). To avoid radiation damage due to cosmic rays, rapid transit or protection by 1m or more of rock would be critical. Low temperatures during interplanetary transit largely stop water chemistry making it a lesser issue with respect to microbial death (and in fact more of a preservation process), so the limiting factor for survival of nucleic acids may be space radiation that is mitigated through time (short transit time to Earth) and shielding (meteorite composition, thickness, etc.).

Meteoritic exchange in the Solar System was 100–1000 times more intense during the heavy bombardment stage 4 billion years ago than it is now. There are signs of numerous fluid flows (Malin and Edgett Reference Malin and Edgett2000a, Reference Malin and Edgett2000b), a possible ancient ocean (Head et al. Reference Head, Hiesinger, Ivanov, Kreslavsky, Pratt and Thomson1999) and sedimentary formations on Mars that suggest a relatively warmer and wetter Mars 3–4 billion years ago. Thus, during the time of extensive meteoritic exchange 4 billion years ago, Earth and Mars had more similar environments, with Mars being much wetter than it is today (Malin and Edgett Reference Malin and Edgett2000a, Reference Malin and Edgett2000b). By the Archean epoch (2.5–4 billion years ago), microbial evolution on Earth may have already proceeded to the point of modern microbial morphologies and enzymatic carbon metabolism with isotopic fractionation (Mojzsis et al. Reference Mojzsis, Krishnamurthy, Arrhenius, Gesteland, Cech and Atkins1999). All known cell-based organisms on Earth share a core of about 500 genes, some or all of which were inherited from a common ancestor. This includes the most conserved of those genes, the small subunit ribosomal RNA gene 16S rRNA in prokaryotes and 18S rRNA in eukaryotes. This common ancestor has been hypothesized to be an archaeal-like hyperthermophile 3–4 billion years ago whose metabolism exploited oxidation/reduction gradients (Pace Reference Pace1991).

Subsequently, the environments on Mars and Earth have diverged: the appearance of oxygen on the Earth 2.5 billion years ago led to the formation of the ozone layer, which decreased UV radiation, while Mars lost its atmosphere as its magnetic field decayed, causing an increase in UV exposure, cooling of the surface and loss of surface water. Extant life on the Martian surface would need to survive temperatures and pressures below the triple point of water, high UV exposure and the oxidizing surface chemistry presumably induced by UV radiation. Despite these extreme conditions, it has been shown that only a thin layer of soil is needed to protect microbes from UV exposure (Cockell et al., Reference Cockell, Catling, Davis, Snook, Kepner, Lee and McKay2000; Mancinelli and Klovstad Reference Mancinelli and Klovstad2000; Schuerger et al. Reference Schuerger, Mancinelli, Kern, Rothschild and McKay2003). In addition, the redox gradient resulting from UV irradiation might actually power microbial metabolism, just as redox gradients in the Earth's crust drive chemolithotrophic metabolism. While there is no doubt that Mars is currently an extreme environment, the adaptability of microbial life on Earth – for example, extremophiles in the driest deserts or coldest Arctic climates – does not make it unreasonable to propose that Martian microbes could have adapted to the gradual decline in water, temperature and UV protection over the past few billion years; and just as the adapted and diverged microbes in Earth extreme environments still bear the signature of their common ancestry in their conserved genes, Martian microbes may also bear theirs as well.

Even if life did transfer between Mars and Earth 4 billion years ago and thrive in the early Martian environment, it may now only thrive in very particular locations (i.e. refugia), for example, deep in the crust where the temperature rises above that of the frozen surface, or at particular volcanic thermal vents. There may also be regions on Mars where liquid water is not in short supply such as the regions near the ice-rich polar ice caps, especially the north cap with its seasonal variation in ice, and perhaps liquid water underneath (Orosei et al. Reference Orosei, Lauro, Pettinelli, Cicchetti, Coradini, Cosciotti, Di Paolo, Flamini, Mattei, Pajola, Soldovieri, Cartacci, Cassenti, Frigeri, Giuppi, Martufi, Masdea, Mitri, Nenna, Noschese, Restano and Seu2018), where it would be expected to have the highest water levels near the surface. In addition, the D/H ratio (deuterium and hydrogen ratio) of water in Martian meteorites suggests a much larger reservoir of water in the crust that is not in equilibrium with the atmosphere (Donahue Reference Donahue1995). This water is predicted to be liquid a few kilometres into the crust, where temperatures rise above surface temperature. In addition, there is evidence for relatively recent Martian volcanic activity, suggesting sources of temperature gradients and fluid flows just below the Martian surface near these sites Malin et al. Reference Malin, McEwen, Carr, Soderblom, Thomas, Danielson, James, Veverka and Hartmann1999. There may also be local hydrothermal systems near the regions of recent volcanic activity (Malin et al. Reference Malin, McEwen, Carr, Soderblom, Thomas, Danielson, James, Veverka and Hartmann1999). InSight lander's seismometer, SEIS, the Seismic Experiment for Interior Structure, detected 174 ‘marsquakes’ over 207 sols, indicating that Mars is seismically active (Giardini et al. Reference Giardini, Lognonné, Banerdt, Pike, Christensen, Ceylan, Clinton, Driel, Stähler, Boese, Garcia, Khan, Panning, Perrin, Banfield, Beucler, Charalambous, Euchner, Horleston, Jacob, Kawamura, Kedar, Mainsant, Scholz, Smrekar, Spiga, Agard, Antonangeli, Barkaoui, Barrett, Combes, Conejero, Daubar, Drilleau, Ferrier, Gabsi, Gudkova, Hurst, Karakostas, King, Knapmeyer, Knapmeyer-Endrun, Llorca-Cejudo, Lucas, Luno, Margerin, McClean, Mimoun, Murdoch, Nimmo, Nonon, Pardo, Rivoldini, Manfredi, Samuel, Schimmel, Stott, Stutzmann, Teanby, Warren, Weber, Wieczorek and Yana2020), however the buildup of massive volcanic cones do not favour active plate tectonics; a key element for hydrothermal vents and the biology that thrives on their energy and nutrient flows.

Martian biological risk assessment: a discussion

The Mars 2020 sample cache will be collected from roughly the first 6–7 cm of the Martian surface, a very harsh, desiccating environment exposed to solar UV radiation. If Martian life is carbon-based, it is likely to be susceptible to sterilization or inactivation by the same technologies used on Earth, because carbon-based Martian life would likely use similar classes of covalent and hydrogen bonds for structural integrity and information storage and retrieval. If it is not carbon-based (e.g. silicon-based), but does use similar classes of covalent and hydrogen bonds for structural integrity, again, Earth-based sterilization/inactivation processes may be sufficient to disrupt molecular structures. If Martian life is another sort of highly robust, exotic type of life – which was not a focus of this set of SWG workshops – it becomes more difficult to predict what a sterilization/inactivation process would look like. This situation raises a critical point regarding placing focus on fundamental chemistries. The SWG felt it reasonable to assume that fundamental chemistries (e.g. covalent and ionic bonds between known molecules in the periodic table) would be present on Mars as they are on Earth. This concept of fundamental chemistries is discussed in more detail subsequently in this paper.

If it can be considered a reasonable model that life on Earth and Mars use similar biochemistries (i.e. nucleic acids, lipids, proteins), then life on Mars would be expected to have specific environmental requirements and similar susceptibilities to biochemical inactivation. Although, life on Earth is enormously diversified into billions of species, these entire cell-based organisms share the common set of 500 genes previously mentioned that are the signatures of life on Earth. Sterilization technologies are extremely effective at inactivating all known pathogens on Earth, including cellular- and non-cellular-based forms, because they share fundamental chemistry. Their mechanisms of action are also known to directly impact chemical bonds in essential structures for life including nucleic acid, protein and lipid structures (McDonnell Reference McDonnell2017). If we then assume that Earth and Mars life follow the same rules due to similar genetic components, then sterilization protocols used on Earth life are likely to work on Martian life.

However, even if such life on Mars is abundant with compatible biochemistry (such as being nucleic acid-based), the likelihood of it being directly pathogenic to humans (or other hosts) is considered very low. Most microorganisms on Earth are not pathogenic, nor harmful, and their abilities to infect host tissues are due to their coevolution between pathogen and host genomes. Microorganisms are usually highly adapted to specific biological niches or hosts, and even when novel pathogenicity arises, as in zoonosis or opportunistic infections, it does not represent a major evolutionary gulf. Emerging human pathogens are often the result of zoonosis in which an existing pathogen moves between related species being modified during this transfer such as coronaviruses, Ebola or HIV which all emerged from other mammalian hosts, or influenza which can transmit from avian or mammalian hosts. Existing microorganisms that coexist with humans over long periods of time can also cause new diseases when the organism takes on new pathogenicity, such as the Escherichia coli strain 0157:H7 that acquired a gene for Shiga toxin, or opportunistically infect a host with a weakened or compromised immune system such as candidiasis yeast infections or Kaposi's sarcoma, a cancer caused by a virus. Certain vector-borne diseases and parasites that have complex life cycles can transmit between disparate parts of the animal kingdom, such as between mosquitos and humans for malaria or yellow fever, or between snails and humans for the trematodes that cause schistosomiasis, with mosquitos and snails sharing a common ancestor with humans an estimated 600–1200 million years ago (Erwin and Davidson Reference Erwin and Davidson2002). Since any putative Martian microorganism would not have experienced long-term evolutionary contact with humans (or other Earth host), the presence of a direct pathogen on Mars is likely to have a near-zero probability.

Other biological risks beyond self-reproducing microorganisms, such as prions, were also considered in the SWG discussions. For example, prions are self-propagating, misfolded proteins with the ability to refold monomeric, analogous proteins that are benign and soluble into insoluble, abnormal, detergent and protease -resistant aggregations that cause harm to biological systems without being classified as alive.

Although prions lack genetic material such as DNA and RNA, they are capable of Darwinian evolution. Like bacteria and viruses, prions have a similar process of mutation and adaptive change (Li et al. Reference Li, Browning, Mahal, Oelschlegel and Weissmann2010). Prions are capable of interspecies transmission (Fernández-Borges et al. Reference Fernández-Borges, Parra, Vidal, Eraña, Sánchez-Martín, de Castro, Elezgarai, Pumarola, Mayoral and Castilla2017) between mammals, mutation development, and through natural selection, the mutations can result in evolutionary adaptations. Prion mutations are linked to changes in prion folding, a process also referred to as conformational change. Mammalian prions adopt several conformations, with each conformation (or strain) capable of precipitating a particular disease; moreover, each specific strain is thought to exhibit distinctive biochemical properties. Some prion strains maintain their unique biochemical signatures, as well as clinical and neuropathological signs, upon transmission to new hosts (Solforosi et al. Reference Solforosi, Milani, Mancini, Clementi and Burioni2013; Das and Zou Reference Das and Zou2016). It is also important to highlight that prions are completely dependent on their hosts for reproduction.

It is most likely that any hypothetical Martian prion or similar protein assembly (if such a prion exists and is present in the cached samples) would be incapable of propagation owing to the lack of available hosts, unless these protein assemblies were essentially similar to human, animal, plant or other Earth analogues, and due to the presence of differing environmental conditions on Earth. Protein folding and functionality depend on temperature, water availability and an appropriate milieus. The conditions on Mars are arid and would not likely promote interaction or propagation of native hypothetical protein assemblies in Earth hosts. Although there is evidence of water vapour in the atmosphere of Mars and past evidence of water activity found in the environment, the low amount of water would likely be insufficient for sustaining such protein functionality.

However, the danger to Earth is not just pathogens. The mandate to ‘…avoid…adverse changes in the environment of the Earth…’ (United Nations 1967) calls into consideration all parts of the Earth's biosphere. The possibility of Martian material disrupting any aspect of the Earth's ecosystem also must be addressed. For example, could Martian material somehow interfere with the ability of photosynthetic bacteria in Earth's ocean to fix carbon dioxide and produce oxygen? Photosynthetic bacteria such as Prochlorococcus are among the most abundant organisms on Earth and intensely important for the health of oxygen-respiring organisms, such as humans and animals. Damage to the ecosystem could be caused by direct cellular infections (i.e. pathogenesis; unlikely for the reasons outlined above), competition for resources, production of biotoxic metabolites or even displacement of organisms, as has been observed with many invasive species of plants, insects and other organisms between regions on Earth. Planetary protection must consider not just human health directly, but the entire biota of Earth.

Organisms evolve to live in a particular environment, and while some are generalists, others can only survive in very specific conditions. In all cases, organisms do not replicate if critical nutritional or environmental conditions are lacking. Invasive species or pathogens have been successful on Earth because they were adapted to similar environmental niches in their original and dispersed ecosystems (Bleuven and Landry Reference Bleuven and Landry2016). There are many described extremophiles that may survive in environments that are extreme to human or animal life (e.g. extremes of temperature or pressure) but do not survive under conditions in our normal habitat (Merino et al. Reference Merino, Aronson, Bojanova, Feyhl-Buska, Wong, Zhang and Giovannelli2019). Thus, it is plausible that any Martian microbe, after it arrives on Earth, would not be viable on Earth due to a lack of its required Martian nutritional and environmental conditions. Indeed, the Martian environment is inhospitable to Earth life, but conversely, the Earth environment is likely to be just as inhospitable to Martian life. Based on these factors, a very low qualitative probability of biological risk can be assumed.

Martian dust and its potential transport to Earth

A thorough understanding of Martian dust and its transport physics is essential to understand the potential biology that could be associated with these particles. Martian dust can originate from various sources on the surface of Mars (Fig. 2): Dust particles can be suspended or wind-carried in the atmosphere; these particles have the potential to fall out onto exposed spacecraft surfaces. On the surface of Mars, dust particles can be found in undisturbed or perturbed regolith such as excavated material. This material can be moved onto exposed spacecraft surfaces during sample tube loading.

Fig. 2. The Mars material of concern – dust is depicted in each yellow highlighted area.

There are now several models in the literature that describe the size distribution and number density of suspended particles in the Martian atmosphere that depend, among others, on location, season and optical depth. Under nominal conditions, a commonly used Martian dust particle distribution is that described by Clancy and Lee (Reference Clancy and Lee1991), which is based on analyses of data from Mariner 9 and Viking. It shows that approximately 95.95% of all suspended particles have a diameter less than 15 micrometers. Data from the Pathfinder and Mars Exploration Rovers (MER) missions shed light on particle accumulation rates. For example, Mars Pathfinder carried a dedicated instrument (Materials Adherence Experiment – MAE) to measure dust deposition which identified the fractional coverage from dust deposition as 0.3–0.4% per sol (Landis and Jenkins Reference Landis and Jenkins2000).

For example, Mars material can accumulate inside a spacecraft or adhere to external surfaces, all of which enable the Martian dust to potentially hitch a ride back to Earth. Particle transport vectors have been derived to track Martian dust particles of concern from the surface of Mars to the surface of Earth. These vectors describe sources and sinks of dust and the process(es) by which it may be transported throughout the journey from Mars to Earth across different environments such as the Martian surface, launch from the surface, achieving Mars orbit, interplanetary transport to Earth, and Earth-reentry and landing. Each environment will expose Martian dust to different conditions. The launch from the Martian surface can strip and heat particles adhered to the external surface of the launch vehicle, and once in orbit, dust particles can be emitted from external spacecraft surfaces. Mars orbit and the interplanetary transfer to Earth exposes particles on the external surface of the spacecraft to solar UV radiation, solar heating, solar ionizing radiation, galactic cosmic rays, extreme temperature (e.g. high, low and fluxes possible), space vacuum and interactive effects of these factors. All of these processes are being studied by the MSR Campaign

An analog of the Martian environment on Earth: the Atacama Desert

An initial dust bioburden estimate is an essential input parameter in developing a microbial reduction protocol. Such a protocol establishes an appropriate verification and validation programme for a given microbial reduction process and allows the user to incorporate the data into a probabilistic risk assessment or other analyses, that may be part of the design and verification processes used by the MSR Campaign. Unfortunately, a Martian bioburden model does not exist, thus an analogue is necessary to satisfy this input parameter. To establish a working model of the BPP potential risk for contamination, biological performance of a proposed Martian bioburden model was developed based on Earth-based analogue environments. The analogue environments focussed on soil and dust vectors since the plausible potential contamination of the return sample would be either from the top layers of the Martian soil, from sampling or surface exposure, or dust in the Martian aeolian environment.

In developing a bioburden model, physics-based, Earth-based, organic-rich maximum bioloads and Earth-based environmental bounding cases were considered. As a worst-case scenario, a physical model could be employed where every particle would be completely comprised of biological material. In this case, the direct volume of dust could be converted into the maximum packaging of biologicals (e.g. cells, viral or proteinaceous particles) based on morphology and their associated calculated volumes. As an example, a spore model could be utilized as depicted in the following equation (Kesavan et al. Reference Kesavan, Schepers, Bottiger and Edmonds2014):

$\rm {{Total\, \, Spore = \lpar Cluster\, \, Diameter\comma\; }\, {m}\rpar ^3}\times {1.2}$

This is considered a worst-case scenario as it would be very unlikely that 100% of the material in any given environment would be biological. Earth-based environments that were organically rich were discussed, but these cases were deemed even more unrealistic since these conditions are not observed in Mars-based science results (Rummel et al. Reference Rummel, Beaty, Jones, Bakermans, Barlow, Boston, Chevrier, Clark, de Vera, Gough, Hallsworth, Head, Hipkin, Kieft, McEwen, Mellon, Mikucki, Nicholson, Omelon, Peterson, Roden, Lollar, Tanaka, Viola and Wray2014). Alternatively, Mars environmental parameters such as desiccation, UV radiation, salinity and temperature were factors used to establish a relevant Earth-based analogue.

To help frame and define this as a worst-case scenario, a biologically driven environment was evaluated to understand the maximum amount of bio-loading based on biotic processes in an Earth organic-rich environment. Organically rich environments that were considered focussed on Earth-soil or dust from agricultural soils. Note that saturated aqueous environments, such as swamps or lake beds, were not considered due to the desiccated nature of the plausible Mars biological sources. The observed ranges of bioloads observed are 10⁸–10⁹ cells g⁻¹ representing 0.01–0.1% of bioparticle loading, assuming the average bacterial cell is 10⁻¹² g (Frossard et al. Reference Frossard, Hammes and Gessner2016). The >99% of non-biological particles directly demonstrates the worst-case end-member of the physics-based model. While a directed Earth-based biological model defines the possibility of biologicals that can be supported on a substrate, it does not consider all the environmental parameters observed on Mars.

When the Mars environmental parameters were assessed, desiccation, salinity, temperature flux and solar UV radiation were the driving conditions noted that could significantly impact microbial survival on Mars. As such, Earth-based environments that exhibited these extremes include the Antarctic Dry Valleys, the Gobi Desert, Sahara Desert, aeolian dust transport phenomena (e.g. Asian and Saharan) and the Atacama Desert. Of all the sites, the most extreme and largest of astrobiological relevance in terms of study and abundance of peer-reviewed data is the Atacama Desert. Culture and non-culture-based cellular enumeration methods are published that evaluated diverse field sites of the Atacama (Navarro-González et al. Reference Navarro-González, Rainey, Molina, Bagaley, Hollen, de la Rosa, Small, Quinn, Grunthaner, Cáceres, Gomez-Silva and McKay2003; Glavin et al. Reference Glavin, Cleaves, Schubert, Aubrey and Bada2004; Drees et al. Reference Drees, Neilson, Betancourt, Quade, Henderson, Pryor and Maier2006; Connon et al. Reference Connon, Lester, Shafaat, Obenhuber and Ponce2007; Lester et al. Reference Lester, Satomi and Ponce2007; Ewing et al. Reference Ewing, Macalady, Warren-Rhodes, McKay and Amundson2008; Lynch et al. Reference Lynch, King, Farìas, Sowell, Vitry and Schmidt2012; Crits-Christoph et al. Reference Crits-Christoph, Robinson, Barnum, Fricke, Davila, Jedynak, McKay and DiRuggiero2013; Idris et al. Reference Idris, Goodfellow, Sanderson, Asenjo and Bull2017; Schulze-Makuch et al. Reference Schulze-Makuch, Wagner, Kounaves, Mangelsdorf, Devine, Vera, Schmitt-Kopplin, Grossart, Parro, Kaupenjohann, Galy, Schneider, Airo, Frösler, Davila, Arens, Cáceres, Cornejo, Carrizo, Dartnell, DiRuggiero, Flury, Ganzert, Gessner, Grathwohl, Guan, Heinz, Hess, Keppler, Maus, McKay, Meckenstock, Montgomery, Oberlin, Probst, Sáenz, Sattler, Schirmack, Sephton, Schloter, Uhl, Valenzuela, Vestergaard, Wörmer and Zamorano2018; Ruginescu et al. Reference Ruginescu, Purcǎrea, Dorador, Lavin, Cojoc, Neagu, Lucaci and Enache2019) and include culture methods (e.g. tryptic soy agar, R2A, plate count agar) to biochemical (e.g. sublimation, phospholipid-derived fatty acids and adenosine triphosphate), direct microscopy and molecular-based techniques (e.g. quantitative polymerase chain reaction). The values of these aforementioned parameters can be utilized to construct a biological cell per gram of soil model to account for multiple variables in the Atacama that include sample depth, rainfall and total carbon-based extrapolations.

Thus, we propose that the hyper-arid regions of the Atacama Desert (e.g. portions of the Yungay) is the most aligned Earth-based extreme environment compared to Mars environmental parameters that have been extensively studied as an astrobiology field site. Importantly, the environmental perturbations on Mars are harsher for microbial life in terms of larger temperature fluxes, lower water availability, more intense solar UVC radiation and the associated growth on highly oxidized substrates. Although there are large differences in environmental conditions in the Atacama Desert compared to the Martian surface, the Atacama remains the most representative Earth analogue that might help establish an upper bound of possible biological limits. Such an Atacama-based model could then be used as a starting point in the design of the MSR campaign's sterilization programme. While the model could provide a cells per gram of soil distribution model, further development and discussion is required into the applicability and utilization of this model as a contamination vector. In particular, the distribution and association of biological material need to be better understood in order to inform the particle-dust analysis and biological models. Aeolian dust analyses have been conducted to evaluate the particulate versus biological-associated particles that can be used. Alternatively, biological particulate loading experiments can be conducted or data can be utilized from aerobiology experiments in Earth's atmosphere to understand particle formation and the fates and transfer processes on Earth for extrapolation to the Mars environment.

Validation of sterilization processes

Sterilization processes are established by performing validation exercises on the equipment and products to be sterilized. There are two primary sterilization validation approaches that exist in industry: an overkill-based method (typically based on using a biological indicator (BI)) and bioburden-based methods

As briefly discussed above, overkill-based methods often use a microorganism that has previously been demonstrated to be highly resistant to a given sterilization process in order to test the efficacy of that process. This efficacy is demonstrated by starting with a known, high count of the test microorganisms (e.g. 10⁶) and determining the degree of inactivation or number of log reductions that occur during a portion of a proposed sterilization cycle. The microorganisms selected tend to be bacterial endospores (e.g. Bacillus spp.), as these typically have high levels of resistance to inactivation (in comparison to other microorganisms). Endospores (henceforth just spores) are also resistant to desiccation and can therefore be made into BIs by inoculating a carrier (e.g. paper, metal) that is stored inside a package that is permeable to the sterilization process. A difficult to sterilize location on the product is determined and can then be challenged by either placing the BI in that location, or if the product location is too small to fit a BI, directly inoculating that location with a liquid suspension of the spores. The exposure time that inactivates all of the spores is called a fractional cycle, for example, a half-cycle exposure. The successful fractional cycle time is then extrapolated (often doubled) to determine the minimum exposure time that will be applied to the products to ensure sterilization. This method is referred to as an ‘overkill’ method, as it is considered a significant overkill based on the assumed starting population and resistance of microorganisms that may be present. Overkill-based methods are traditionally employed for ethylene oxide (EO), dry heat, moist heat (steam) and other gaseous sterilization methods such as hydrogen peroxide, nitrogen dioxide, chlorine dioxide, ozone and others.

Bioburden-based methods by contrast require a more detailed knowledge of the naturally-occurring bioburden (the population of viable or detectable microorganisms) on the product. The resistance of this bioburden and the extent of the sterilization cycle required are then determined in one of two ways:

Option 1: Using a sterilization table to determine the sterilization cycle based on the product's bioburden count. The table provides a fractional sterilization cycle that is applied and followed by a sterility test. If the sterility test gives acceptable results, the sterilization table provides the complete sterilization cycle to be applied to the product.

Option 2: Determining the sterilization cycle using an incremental series of sterilization exposures followed by sterility tests. The results of the incremental sterilization exposures and sterility tests are used to calculate the complete sterilization cycle to be applied to the product.

Both bioburden-based options provide the ability to quantify the required SAL. Bioburden-based methods are most commonly applied only to radiation sterilization, but may also be applied to other processes. Although bioburden-based methods may not include the same level of overkill as the overkill-based methods, they both are considered equally safe regarding potential impacts to a patient. Combinations of these methods can also be considered as validation approaches.

Overall, both overkill- and bioburden-based methods of sterilization validation require some estimation of the types, levels and resistance of bioburdens that may be found on products. For samples coming from Mars, assumptions based on data will need to be made about potential types of viable Martian material, their quantities and the extent of the inactivation processes in order to assign a safety margin to the collection of sterilization procedures.

Sterilization based on fundamental chemistries of life

It is possible to imagine unique and putative harmful agents on Mars that are both resistant to all forms of inactivation and that are pathogenic or hazardous to life on Earth. However, all available knowledge on the topic, including the previous portions of this paper, point to this potential scenario being qualitatively highly improbable. To date, all available knowledge indicates that the fundamental chemistries of life that occur on Earth (e.g. covalent, ionic and hydrogen bonds between molecules) are also in place on Mars. There is no evidence of indigenous, advanced life on Mars, despite many orbiting and surface missions to the planet, but it is not clear if microbial life or chemical biomarkers are present. As we assess the potential hazards of Martian life (if present) to terrestrial Earth life, it was agreed upon by the SWG that focussing on inactivation of the fundamental chemistries is appropriate.

Earth biological challenge agents can be used to validate the efficacy of sterilization and inactivation modalities. However, emphasis should also be placed on the modes of action of these technologies and particularly their effects on the fundamental chemical properties (e.g. the assembly and function of the macromolecules that make up the various forms of known life). For example, although it might not make sense to focus all sterilization efforts specifically on DNA or RNA inactivation or damage, as those configurations may not be found on the Martian surface; it is sensible to focus on nucleic acids in general and the bonds that hold them together and how they might be disrupted. Thus, the term nucleic acid is used in this paper to represent DNA/RNA-type materials and the inactivation that occurs to those materials. Likewise, it is not reasonable to focus other efforts specifically on the prions that cause transmissible spongiform encephalopathies such as Creutzfeldt-Jakob Disease in humans (as they are entirely based on misfolded human or animal proteins), but it is reasonable to focus on inactivation methods that affect protein assemblies similar to prions and the means of fully denaturing (inactivating) those protein polypeptides. Thus, the term protein assembly is used in this paper to represent prion-type materials and the inactivation that occurs to those materials. For nucleic acids, the primary bonds in place are covalent (between the bases, sugars and phosphate groups of each strand) and hydrogen (between the two strands). For prion-type protein assemblies, the primary bonds in place are peptide, hydrogen and disulfide bonds. Additionally, there are interactions involved with protein folding that include hydrophobic and electrostatic interactions as well as van der Waals forces involved in the polypeptide main chain and amino acid residues to create a given secondary assembly that is associated with the transmissible and disease-causing form of the protein. If inactivation processes engineered into the MSR system can disrupt those types of bonds and interactions, the SWG agreed that it should be sufficient to disrupt those same bonds between other, similar chemistries potentially found in Martian material. Therefore, much of the general focus should be on inactivation of the fundamental chemical bonds of nucleic acid and protein assemblies.

The additional types of biological challenge agents that are intrinsic in the basic requirements for sterilization methods are viruses. Viruses can range in resistance to inactivation, with non-enveloped viruses well established to be the most resistant forms (Eterpi et al. Reference Eterpi, McDonnell and Thomas2009) but less resistant than other forms of microorganisms such as bacterial spores (McDonnell Reference McDonnell2017). It is well understood that with some methods of inactivation, such as chemical or heat, bacteria that form dormant spores, such as Bacillus or Clostridium species, represent a much greater challenge to inactivation than viruses, whether the viruses are enveloped or non-enveloped viruses or bacteriophages (U.S. Food and Drug Administration 2020). For radiation-based inactivation methods including UV (Rockey et al. Reference Rockey, Young, Kohn, Pecson, Wobus, Raskin and Wigginton2020), as the main mechanism of action is known to be DNA or RNA damage, protein structures and prions lack associated DNA and RNA molecules, and therefore prions could present the greatest inactivation challenge.

When considering appropriate Earth analogues for determining inactivation protocols, it must be addressed from two different perspectives:

1. (1) Nucleic acid based: spores of bacteria that are resistant to chemical- or heat-based inactivation methods can be prioritized as BIs as they represent a more difficult class of organism to inactivate compared to other types of Earth microorganisms and viruses.

2. (2) Protein based: prion-type proteins are naturally resistant to heat-based inactivation methods and can be prioritized as general-process indicators as they represent a more difficult class of material to inactivate compared to viable microorganisms.

Concept of microbiological safety

The United States Food and Drug Administration (US FDA), and similar regulatory agencies worldwide, require health care manufacturers (e.g. medical device and pharmaceutical manufacturers) to demonstrate that their products are safe and effective. The level of safety with health care products is rarely, if ever, so high that the potential risk to a patient is absolutely zero. Nevertheless, if adequate controls are maintained and monitored for effectiveness, the level of risk to patients can be reduced to acceptable levels.

As discussed earlier, in the application of sterilization processes, the concept of safety is largely addressed by applying a SAL to the diverse processes. In some instances, different levels of sterility assurance other than 10⁻⁶ are permissible based on an assessment of risk. For example, a SAL of 10⁻³, which corresponds to a one in 1000 probability of a viable microorganism being present, is appropriate, such as traditionally in the USA for sterilization of drapes to cover equipment in an operating room during surgery. Essentially, these and other SALs can be shown to be accepted based on a risk assessment and remembering that the definition of ‘sterile’ is being free of living microorganisms and not necessarily a theoretical mathematical extrapolation. Additionally, documents are available (International Organization for Standardization 2017a, 2019) that provide the concepts for justification of alternative SALs based on a thorough risk assessment and the inability of products to withstand typical sterilization processes. Thus, in the healthcare industry, flexibility regarding the extent of sterilization is provided depending on the situation and the output of risk assessment. The MSR SWG has determined that this concept of microbiological ‘safety’ and use of risk assessment is reasonable and appropriate when discussing the level of inactivation required for uncontained material on spacecraft surfaces. As is the case with health care products, the potential risk to humans and planet Earth cannot be described as non-existent or zero. However, with a thorough assessment of what is known and through making reasonable assumptions based on Earth empirical data, an understanding of the potential risk can be determined and quantified.

Biological challenge agents

One of the SWG conclusions from discussions on Mars life was that it is reasonable to employ Earth-based surrogates to challenge potential inactivation processes. This is due, in part, to the possibility that a common ancestry between life of Mars and on Earth may be plausible, or at the very least that the planets use the same fundamental chemistries.

A combination of biological reduction steps including passive inactivation due to environmental conditions during space travel (e.g. solar ionizing radiation and UV, deep space vacuum, solar heating, etc.) and active inactivation spacecraft systems (e.g. chemicals, heat, spacecraft-induced UV radiation) should be considered to understand and reduce the viability of potentially uncontained Martian material through the entire mission architecture from Mars orbit to arrival on Earth. To validate these processes, a programme should be initiated to establish the biological challenges, coupons and test protocols for biological reduction assurance and to establish expected levels of inactivation (i.e. D-values, or the time or dose required to achieve inactivation of 90% of a population of the test microorganisms) and/or the impact of inactivation process variables (e.g. for heat inactivation, Z-values or the change in temperature of a thermal sterilization process that produces a tenfold change in D-values) for both single modalities as well as the additive impacts from multiple modalities. The standardization of a common test programme was considered paramount, as multiple labs could conduct testing and be used for third party verification.

A wide approach to gain expert input for selecting biological challenges was conducted, by surveying the scientific community for the largest spectrum of extreme environmental isolates to include bacterial spores, fungi, archaea, plasmids and prions. The preliminary polling consisted of environmental microbiology, astrobiology, planetary protection and sterilization experts from academia, industry and government labs worldwide. The polling identified 75 candidates, and 13 candidates were down selected to begin initial testing. The candidates that were considered consisted of preferential biosafety level 1 microorganisms that are relevant to spacecraft, the space environment, industrial standards, and that have growth conditions that favour standard microbiology lab resources (e.g. standard doubling times, temperatures, pressures, etc.). After discussing this approach with the SWG, further inputs into accessing the sterilization community was a resulting action. A subset of the 2019 Kilmer Conference attendees was polled. The approach was also presented at the US Environmental Protection Agency's 2019 International Decontamination Research and Development Conference (Benardini and Smith, Reference Benardini and Smith2019). Feedback from both of these communities was utilized. In the end, 39 labs were polled resulting in 101 candidates and 17 candidates were then down-selected (Table 1). Spore-forming bacteria of known high-level resistance to inactivation were selected to be the first candidates for testing due to their hardy nature, ease of growth and viability on test surfaces when desiccated to facilitate ease of shipping and testing.

Table 1. Proposed biological challenge agent list

Defined bacterial spore stocks and a standard set of test surfaces (coupons) were suggested for procurement so that all test laboratories can use the same standardized spore crop, starting population and associated coupon material of a known cleaning level and surface finish. The coupons will be seeded with a target population of approximately 2 × 10⁶ spores and individually packaged for enumeration prior to and after each test procedure to ensure the target populations and viability of the spores on all coupons. Further protocols to prepare test coupons should also be explored for the non-spore-forming microorganisms as identified future work.

It is not clear that typical spore-forming microorganisms would provide sufficient assurance that an inactivation process will be effective without prior knowledge of the nature of life on Mars, if present. Challenge microorganisms or BIs traditionally used at NASA have been identified by past mission requirements that mainly focussed on spore-forming bacterial species. Prions have proved in the past to be highly resistant to traditional sterilization modalities such as EO and radiation that are usually sufficient for inactivating nucleic acid-containing agents (McDonnell and Comoy Reference McDonnell, Comoy and Walker2020; McDonnell Reference McDonnell and Walker2013a, Reference McDonnell, Fraise, Lambert and Maillard2013b). The SWG suggested that tests be performed to evaluate the effectiveness of dry heat on protein assemblies, while concurrent trade-off studies could be performed with other sterilization modalities.

Sup35, a non-pathogenic yeast strain from Saccharomyces cerevisiae, was identified (Table I) to have amyloid-based prions that can be used as an acceptable surrogate for inactivation testing (Chernoff Reference Chernoff2007). Yeast proteins have been successfully employed by researchers to understand fundamental prion properties such as aggregation and self-propagation (Chernova et al. Reference Chernova, Wilkinson and Chernoff2017). At a molecular level, the yeast prion protein selected for testing has been extensively characterized and exhibits aggregation behaviour similar to mammalian prion protein (PrPsc) (Chernova et al. Reference Chernova, Wilkinson and Chernoff2017; Wilson et al. Reference Wilson, Bommarius, Champion, Chernoff, Lynn, Paravastu, Liang, Hsieh and Heemstra2018). Yeast aggregating proteins, despite also being termed ‘prions’, are not known to be dangerous to humans, due to the species barrier, and can be used as a cost-effective and safer alternative to working with mammalian prions (Chernova et al. Reference Chernova, Chernoff and Wilkinson2019).

While there is much literature to suggest how bacterial spores are sterilized, there remains some knowledge gaps in the accurate molecular mechanisms of prion propagation and inactivation. The stabilization of the core assembly, as previously mentioned, is supported by hydrogen bonds and other interactions involving the polypeptide main chain and amino acid residues. Thus, inactivation data should be gathered regarding the prion protein's stability and disruption. Heat will likely increase the kinetic energy, causing molecules to vibrate so rapidly that the peptide bonds and non-polar hydrophobic interactions are disrupted in the process. Testing will require prion proteins to be aggregated and heat exposed, followed by standardized protein tests including sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis, which could be used to visualize the protein conformational changes based on the presence or absence of aggregated and monomeric fractions (Chernoff et al. Reference Chernoff, Uptain and Lindquist2002). Detection of conformational changes do not guarantee the absence of small fractions of the proteins retaining the active prion conformations, and thus, additional assays will likely be required to demonstrate complete degradation of the prion proteins into non-functional forms. A fully degraded/inactivated prion protein is unable to renature or restore pathogenicity by refolding. Thus, prion inactivation assays should be developed to determine if the treated aggregate samples with the proposed inactivation protocols have the ability to seed conformational changes and aggregation with the addition of fresh monomeric subunits. Additional yeast prion tests should be performed to understand the general biochemistry of how prions respond to stressors (i.e. heat, UV, vacuum) relevant to Mars samples, and which specific bonds are being disrupted as a result of the processes.

Test procedures for all biological challenge agents should include sterilization industry best practices such as relevant portions of the international standard series ISO 11138 (American National Standard Institute/Association for the Advancement of Medical Instrumentation 2019, 2017) ‘Sterilization of Health Care Products – Biological Indicators’ and should be performed on representative spacecraft materials. Test procedures should also include previous NASA methodologies to determine D-values using thermal spore exposure vessels (Kempf et al. Reference Kempf, Schubert and Beaudet2008; Schubert and Beaudet Reference Schubert and Beaudet2011) or equivalent. Multiple labs would then test representative BIs to establish inactivation values (D- and/or Z-values) by analysis of sterilization/inactivation curves.

UV radiation sterilization on Mars and during Mars sample return

By Earth's standards, Mars is a harsh and inhospitable environment. The Martian atmosphere is dominated by the presence of CO₂, but also contains, in much lower amounts, O₂, Ar, CO, N₂ and H⁺ (Chevrefils et al. Reference Chevrefils, Caron, Wright, Sakamoto, Payment, Barbeau and Cairns2006). The Martian surface is highly oxidizing, oligotrophic, and is exposed to high levels of solar ultraviolet radiation (UV). The climate of Mars is cold, dry and Martian dust storms occur frequently that are immense in size and can be highly destructive. Although most Earth-borne microorganisms would perish quickly under these conditions, any putative Martian microorganism that exists at the surface would have either evolved the necessary environmental resistance mechanisms to adapt and survive close to the surface or would be surviving in deeper or more sheltered niches. While the entirety of our knowledge of biology is based on life as we know it on Earth, that knowledge can still yield critical insights into how potential extant Martian microbiota may live in such an inhospitable environment, and what challenges and risks it may pose by introducing such an organism into Earth's biosphere. If we can apply any of our knowledge of life as we know it, to life as it could have evolved on another planet such as Mars, then the SWG agreed we might reasonably make the argument that there could be organisms on, or under, the surface of Mars that have evolved adaptations to persist within its harsh, yet not necessarily inhospitable, UV-exposed environment.

Ultraviolet radiation is an effective microbial reduction modality. Few Earth microbes can withstand more than a few minutes of exposure of UVC (200–280 nm); however, some microbial organisms such as Bacillus pumilus SAFR-032 display an ability to withstand much higher doses of UVC (up to 4 kJ/m²) than any other organisms on Earth (Schuerger et al. Reference Schuerger, Richards, Newcombe and Venkateswaran2006; Osman et al. Reference Osman, Peeters, La Duc, Mancinelli, Ehrenfreund and Venkateswaran2008). UV-resistant microbes have developed evolutionary adaptations which allow them to withstand much higher levels of UV. Studies have shown that while initial exposures to UV can be effective in killing a high proportion of B. pumilus SAFR-032 within the exposed populations on spacecraft surfaces (Schuerger et al. Reference Schuerger, Richards, Newcombe and Venkateswaran2006), a small proportion of a population can survive in protected pits, crevices or shaded niches (Moores et al. Reference Moores, Smith, Tanner, Schuerger and Venkateswaran2007). This ability to survive seemingly lethal doses of UV exposures has been attributed to a number of unique physiological mechanisms relating to UV resistance such as those responsible for the repair of UV-damaged DNA (Chatterjee and Walker Reference Chatterjee and Walker2017).

Arguments have been made that a hypothetical Martian microbiota would be no more resistant to UV than an Earth microbe. However, this view does not fully encapsulate the questions surrounding the ability of UV to penetrate into the Martian subsurface, which is dependent on the radiation energy, the surface geochemistry and particle size distribution of the fines.

There is evidence that between 0.5 and 2 mm of contiguous dust is required to completely shield an organism from being inactivated by solar UV on Mars (Link et al. Reference Link, Sawyer, Venkateswaran and Nicholson2004; Schuerger et al. Reference Schuerger, Richards, Newcombe and Venkateswaran2006). At the microbial scale, there are almost an infinite number of niche environments on the surface of Mars which could provide a complete respite from solar UV. This shielding effect may also be applicable on the outside of spacecraft surfaces if an organism finds itself encapsulated or otherwise shielded within a thin layer of Martian dust. In this respect, UV could potentially reduce a bioload on spacecraft surfaces while still in the Martian atmosphere, but ultimately fall short of complete sterilization. The primary focus of the UV sterilization discussions during the SWG workshops was to present the current foundational knowledge relating to UV's potential to sterilize a hypothetical Martian organism.

The solar UV exposures may also affect the assembly and prion activity. Prions are known to be resistant to UVC (200–280 nm) as typically used on Earth for disinfection applications; however, it was highlighted at the workshops that broad-spectrum UV or other radiation exposures analogous to that on Mars have not been tested on protein assemblies such as prions. Since breaking peptide and hydrogen bonds as well as hydrophobic and electrostatic interactions and van der Waal forces is necessary for prion inactivation, further research is needed to determine UV susceptibility at wavelengths more appropriate to those bonds and forces. Understanding environmental conditions on Mars may provide insights to the challenges potential Martian protein assemblies may have to overcome to retain functionality similar to that observed on Earth.

At the first meeting in January 2019, two primary objectives regarding UV sterilization were presented to the members of the working group.

1. (a) To investigate the sterilization potential of Mars UV on dust that might accumulate on the exterior surfaces of MSR spacecraft.

2. (b) To investigate the sterilization potential of the Mars-to-Earth cruise environment on dust that might be present on exterior spacecraft surfaces.

The sterilization potential of Mars UV on dust

The absence of ozone in the Mars atmosphere results in a broader UVC spectrum at the Martian surface. Exposure to this solar radiation is from direct, diffuse and reflected UV radiation. There are a variety of factors to consider in order to understand the degree of exposure received by a given surface. Factors which influence UV exposure include:

• • Structure and composition of the Martian atmosphere

• • Optical properties of Mars Aeolian and surface dust particles

• • Solar flux

• • Spatial (geographic location) and temporal (day of year, local time, etc.) heterogeneities

• • UV particle contact time

• • Spacecraft orientation

• • Spacecraft geometry

An underlying assumption when considering the UV sterilization potential within the Mars atmosphere is that the damaging effects of UV exposure on hypothetical Mars microorganisms is comparable to that which occurs in Earth microorganisms. This is the basis for understanding Mars UV within the framework of ‘life as we know it’ and is consistent with previous discussions of fundamental chemistries.

In 2017, an exhaustive review of peer-reviewed studies was performed at JPL spanning 50+ years of research on UV inactivation of microbial species (Fig. 3). This work was performed primarily to compile UV lethality data to serve as inputs to models being examined for the Mars 2020 mission.

Fig. 3. UV inactivation of microbial species.

Data from UV measurements from NASA's Mars Science Laboratory (MSL)/Rover Environmental Monitoring Station (REMS) and Pathfinder missions indicate that a microbial population exposed to Mars UV would be reduced by 99.999% in only a few minutes after sunrise (Cockell et al., Reference Cockell, Catling, Davis, Snook, Kepner, Lee and McKay2000; Schuerger et al. Reference Schuerger, Mancinelli, Kern, Rothschild and McKay2003; Gómez-Elvira et al., Reference Gómez-Elvira, Armiens, Carrasco, Genzer, Gómez, Haberle, Hamilton, Harri, Kahanpää, Kemppinen, Lepinette, Soler, Martín-Torres, Martínez-Frías, Mischna, Mora, Navarro, Newman, de Pablo, Peinado, Polkko, Rafkin, Ramos, Rennó, Richardson, Rodríguez-Manfredi, Romeral Planelló, Sebastián, Juárez, Torres, Urquí, Vasavada, Verdasca and Zorzano2014). This is the result of a dose of over 12 kJ h⁻¹ of UVC (at local noon on the equator; Cockell et al. Reference Cockell, Catling, Davis, Snook, Kepner, Lee and McKay2000; Patel et al. Reference Patel, Zarnecki and Catling2002; Schuerger et al. Reference Schuerger, Mancinelli, Kern, Rothschild and McKay2003) which is equivalent to the UV required to kill the most UV-resistant microorganism known here on Earth. However, in order to assess the full sterilization potential of Mars UV, the degree of shading and shielding must also be considered. Dust deposition, whether on the surface of Mars or on the spacecraft, can provide spores with protection from UV exposure, and the degree of protection is dependent on the thickness of the dust layer (Mancinelli and Klovstad Reference Mancinelli and Klovstad2000; Schuerger et al. Reference Schuerger, Golden and Ming2012). Several other studies have concluded that both airborne and surface dust loading is an important component in the attenuation and sterilization potential of incoming solar UV in the Martian atmosphere (Schuerger et al. Reference Schuerger, Mancinelli, Kern, Rothschild and McKay2003; Newcombe et al. Reference Newcombe, Schuerger, Benardini, Dickinson, Tanner and Venkateswaran2005; Richards et al. Reference Richards, Newcombe and Venkateswaran2006; Osman et al. Reference Osman, Peeters, La Duc, Mancinelli, Ehrenfreund and Venkateswaran2008; Vaishampayan et al. Reference Vaishampayan, Rabbow, Horneck and Venkateswaran2012). Additionally, life can escape the lethal effects of UV by ‘hiding’ in pits, crevices, etc. (Moores et al. Reference Moores, Smith, Tanner, Schuerger and Venkateswaran2007).

The sterilization potential of Mars–Earth cruise environment on dust

The interplanetary cruise-phase environment between Mars and Earth is composed of several factors that influence microbial survival including:

• • High vacuum

• • Severe desiccating conditions

• • Extreme temperature fluctuations

• • Solar UV radiation

• • Solar particle events

• • Galactic cosmic rays

Of these factors, solar UV radiation and solar heating appear to be the leading contributors limiting microbial survivability on external spacecraft surfaces. However, some studies indicate that some microorganisms may survive these cruise conditions if solar heating and UV are removed (Horneck et al. Reference Horneck, Moeller, Thierry, Mancinelli, Nicholson, Panitz, Rabbow, Rettberg, Spry, Stackebrandt, Vaishampayan and Venkateswaran2012). Additional information needs to be obtained to better understand the effects of the Mars–Earth cruise environment on spacecraft-associated microorganisms.

For the second SWG workshop, the primary focus of the UV presentation centred on whether a consensus could be reached by the SWG on whether Mars UV and Mars-Cruise UV could reasonably provide some degree of sterilization. To frame the discussion, four propositions were presented to the SWG:

1. (1) UV is highly effective at killing microorganisms.

2. (2) Any extant Martian life would exhibit a range of UV resistance (from no resistance to high), but none are expected to exhibit absolute immunity to UV. Evidence presented to support this proposition included:

   1. (a) In Mars analogue environments here on Earth, bacteria do not generally relegate themselves to life on the surface where environmental perturbations are at their greatest; microbiota seek refuge in environmental niches which provide protection from the harshest environmental stressors (Dartnell Reference Dartnell2011).

   2. (b) There is no nucleic acid-based life, as we know it, capable of withstanding UV radiation at doses equal to that which occur on the surface of Mars, for more than a few hours. Following are quotes that lend credence to this statement:

      ‘Although the various Bacillus spp. exhibited diverse levels of UV resistance, none were immune to UV irradiation, and, thus, all species would be expected to be inactivated on Sun-exposed spacecraft surfaces within a few tens-of-minutes to a few hours on sol 1 under clear-sky conditions on equatorial Mars. We have not observed any phenomena in these experiments nor have we found any literature that would support the conclusion that common terrestrial microorganisms typically found on spacecraft are “immune” to the UV environment on Mars.’ (Schuerger et al. Reference Schuerger, Richards, Newcombe and Venkateswaran2006)

      ‘UV radiation was the key parameter that determined survivability of spores under simulated Martian conditions; direct exposure to UV radiation resulted in rapid and nearly complete inactivation of microbial cultures.’ (Nicholson et al. Reference Nicholson, Schuerger and Setlow2005)

      ‘UV reaching the surface of Mars has a 1000-fold greater biocidal effect than on Earth.’ (Rummel et al. Reference Rummel, Beaty, Jones, Bakermans, Barlow, Boston, Chevrier, Clark, de Vera, Gough, Hallsworth, Head, Hipkin, Kieft, McEwen, Mellon, Mikucki, Nicholson, Omelon, Peterson, Roden, Lollar, Tanaka, Viola and Wray2014)

3. (3) In addition to the bioburden reductions resulting from naturally occurring solar UV exposures, artificial (i.e. engineered) UV sources could potentially be developed and deployed to result in improved cumulative inactivation kinetics.

4. (4) Knowledge and data gaps exist which limit our ability to calculate the degree of bioburden reduction resulting from UV exposure. Needed items to fill these gaps include:

   1. (a) Laboratory testing and experimentation for solar and artificial UV exposures to a wider microbial species diversity than is heretofore been available in the literature.

   2. (b) UV modelling specifically relevant to the future MSR architecture missions.

   3. (c) Dust modelling on Mars.