3

Considerations for Reducing Bioburden Requirements

The presence of harmful contamination depends on the likelihood that terrestrial biota can (1) be delivered, (2) survive, (3) proliferate, and/or (4) be transported away (either laterally or with depth) from a landing site and, as a result, creates the risk of confounding a future search-for-life experiment on Mars. The committee bases its assessment of criteria for permitting some missions to land with relaxed spacecraft bioburden requirements on assessment of the contamination risk for each mission and landing site. Missions whose objectives include the search for evidence of Martian life (i.e., Category IVb missions) will use more rigid contamination controls than the committee needs to consider, and so such missions are not addressed here. Control of the likelihood of delivery of terrestrial biota to Mars depends on the approaches and rigor of decontamination and/or bio-assays that are employed on a mission (discussed in Chapter 4). First, the committee focuses on risk factors (2) through (4)—that is, survival, proliferation, and transport. Then the committee focuses on Special Regions on Mars, leading into the committee’s findings with respect to the principal elements of the study charge—the criteria for landing sites with relaxed spacecraft bioburdens, means to validate that the criteria can be met, and examples of potentially acceptable sites (p. 40). Finally, the committee outlines an approach to implementing reduced bioburden requirements (p. 45).

CONDITIONS FOR SURVIVAL, PROLIFERATION, AND TRANSPORT OF TERRESTRIAL BIOTA ON MARS

Background

In recent decades, orbiters, landers, and rovers have provided abundant evidence that Mars once had a sporadically or persistently wetter climate and may have once been habitable. Mars may continue to be a habitable planet, in spite of its loss of atmosphere and liquid water resulting in a present surface environment hostile to Earth organisms. Following a watery period billions of years ago, Martian life, if it existed, may have become subterranean and/or dormant, or it may have become extinct.

Microorganisms can exist in a number of metabolic states:

• Growth, in which cells are actively dividing;
• Maintenance, in which cells remain active and conduct measurable catabolic and/or anabolic processes but are not actively dividing;
• Dormancy, a reversible state including spore forms, in which cells are not measurably metabolizing, but may be conducting maintenance activities;
• Deceased, in which cells are neither replication-proficient nor capable of becoming metabolically active, but may still contain intact biomolecules (DNA, proteins and lipids) (Blazewicz 2013).

TABLE 3.1 Metabolic Categories

Any organism in the states of growth, maintenance, or dormancy is considered viable.

The combined energetic, nutrient, temperature, and water requirements for the maintenance of, or transition between, any of these states are not well-understood in terrestrial microorganisms. However, the committee focuses its discussion on what is known about the following Mars-relevant conditions likely to influence microbial survival or proliferation:

• Temperature,
• Liquid water,
• Ultraviolet C (UVC, or short-wavelength ultraviolet light, from 200-280 nm)¹ and galactic cosmic rays (GCRs, high-energy particles, chiefly protons), and
• Winds and other means of transport.

While the committee acknowledges that interactions between these conditions may lead to complicated physiological outcomes, the committee treats these individually for clarity of discussion.

In addition to a physically favorable habitat, life requires a renewable supply of nutrients and other essential growth factors, including one or more sources of energy, electrons, and carbon. Depending upon how these requirements are met, organisms can be classified into several metabolic categories (Table 3.1; Karl 2007) or into more than one (“mixotrophy”). Energy sources include sunlight (termed photo-) and chemical (termed chemo-). In certain deep lithosphere environments on Earth, and possibly in analogue Martian habitats, radioactive decay (radio-) could provide an alternative source of energy to support microbial life. Sources of electrons can derive from either inorganic (termed -litho-; e.g., ammonium, nitrite, hydrogen, thiosulfate, and sulfide) or organic (termed -organo-) compounds (Table 3.1). Finally, carbon can be derived from inorganic (-autotroph) or organic (-heterotroph) substrates.

Specific metabolic designations are assigned based on these requirements. For example, photosynthetic organisms like cyanobacteria are photolithoautotrophs because they derive their energy from sunlight, their electrons from water, and their carbon from carbon dioxide. In contrast, most bacteria are chemoorganoheterotrophs because all three major requirements are derived from reduced organic matter (e.g., glucose, amino acids, or other organic molecules). Obligate photolithoautotrophy requires only sunlight and inorganic substrates, and so these organisms are often termed autotrophs (self-feeders), in contrast to most other life forms that are termed heterotrophs. According to the Oparin-Haldane theory for the origin of life on Earth, the prebiotic synthesis of organic matter on Earth most likely initially promoted heterotrophic growth, with oxygenic photolithoautotrophy appearing nearly 1 billion years after the appearance of the earliest forms of microbial life. Martian life forms, extant or extinct, may have also arisen from what some have called “soup of organics” although alternative theories for the origin of life, such as the evolution of autotrophy catalyzed by mineral surfaces (the autotrophic theory [Wachtershauser 1988]), are also possible.

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¹ Some UVB is relevant, but UVC dominates and will be referred to as UVC throughout the rest of the report. The committee considers UVA and UVB to be far less bacteriocidal than UVC.

In addition to the requirements discussed above, atmospheric composition should be considered in models of microbial growth and survival. Based on data collected by the Curiosity rover, the annual mean composition of the Martian atmosphere is 95.1 percent carbon dioxide (CO₂), 2.6 percent nitrogen (N₂), 1.9 percent argon (Ar), and <0.2 percent oxygen (O₂), with observed seasonal variations of 1, 10, 9.7, and 13 percent for CO₂, N₂, Ar, and O₂, respectively (Trainer et al. 2019). This large percentage contribution of CO₂, compared to Earth’s atmosphere (~0.04 percent), may preclude the survival and proliferation of some terrestrial microorganisms. Furthermore, because the average atmospheric pressure on Mars is <1 percent that on Earth, the effective concentration of O₂ in the Martian atmosphere is less than 0.01 percent, and would not support the growth of known aerobic (oxygen-requiring) terrestrial microorganisms. There are additional atmospheric factors such as diurnal O₃ changes and free radicals, but the committee found that the UV environment dominates the effect on survivability, and therefore those effects are not considered here in detail.

Survival of Biota on the Martian Surface and Subsurface

The microbial metabolic states described above may be further divided into states that are characteristic of survival, and states that are characteristic of proliferation. The distinction is important in the assessment of risk for planetary protection, because harmful contamination is most likely to arise as a result of proliferation (see p. 25). Here, survival encompasses cells in states of maintenance or dormancy. For the purposes of this report, unless explicitly defined otherwise, the committee adopts the following definitions: uppermost subsurface as the upper 10 cm, shallow subsurface as the upper 1m, and deep subsurface as deeper than 10 m.

Temperature

Although organisms can survive in a frozen or semi-frozen state and may be metabolically active at −40°C (233 K) (Price and Sowers 2004), prevailing surface temperatures on Mars (average ~210 K, −63°C) are generally incompatible with the proliferation of life as we know it (see p. 25). At the surface and shallow subsurface temperatures of Mars, the risk of harmful contamination is negligible, unless a contaminated environment is connected to other habitable sites. In this context, terrestrial microorganisms that survive in extreme environments on Earth serve as proxies for contaminating microorganisms on Mars; bacteria have been recovered and revived from the interior of the Antarctic Ice Sheet (Christner et al. 2006; Karl et al. 1999; Priscu et al. 1999), where temperatures may be less than −50°C (223 K) (Macelloni et al. 2019), suggesting that survival is possible at even lower temperatures. However, the metabolic state of cells in situ is unknown. Notably, organisms deposited on Antarctic ice sheets are not strictly extremophiles, but are broadly sourced via atmospheric transport, suggesting that a broad range of microorganisms can survive within ice matrices (Santibáñez et al. 2018). Microorganisms are routinely preserved in Earth laboratories in liquid nitrogen (−196°C, 77 K), and the addition of chaotropic substances, in spite of their generally disordering effect on macromolecular systems, can expand the temperature window in which microorganisms can survive (Chin et al. 2010). Some eukaryotes are also freeze-tolerant; for example, the Antarctic nematode Panagrolaimus davidi can survive freezing of all of its body water and resume metabolic activity on thaw (Wharton and Ferns 1995). Together, these examples highlight the ability of diverse forms of life to survive at low temperature. There is no evidence for a lower temperature limit for the existence, or preservation, of life, and temperature alone is not expected to limit the survival of microorganisms on Mars.

Desiccation

The low atmospheric pressure on the surface of Mars is highly desiccating. Life as it is understood is based on aqueous chemical reactions; therefore liquid water is the solvent for life. Similar to the growth-limiting effects of low temperature, drying alone will prevent cell proliferation, and this further reduces the risk of harmful contamination. Desiccation, like freezing, is highly preservative, which increases the risk of viable cell transport to potentially interconnected habitable aqueous environments. Under conditions of extreme desiccation, many unicellular and even some multicellular organisms can enter a state of cryptobiosis, in which intracellular water is lost and metabolism is undetectable. As an example, invertebrate animals such as tardigrades, rotifers, and nematodes are extremely resistant to desiccation and can survive in a cryptobiotic state for extended periods of time (e.g., years to decades for tardigrades [Hibshman et al. 2020]). Bacteria and Archaea also possess a number of strategies that allow them to survive desiccation, including sporulation, the accumulation of sugars and small-molecule Mn antioxidants, and the formation of biofilms (Anderson et al. 2012; Frösler et al. 2017; Sharma et al. 2017; Laskowska and Kuczyńska-Wiśnik 2020; Bruckbauer and Cox 2021). Based on current understanding, desiccation alone is not expected to limit the survival of all terrestrial microbial life.

Radiation: UVC and GCR

Across the tree of life, there is a close relationship between cellular desiccation tolerance and radiation resistance. Prokaryotes and simple eukaryotes that are desiccation-resistant are typically also resistant to UVC and ionizing radiation (Mattimore and Battista 1996; Slade and Radman 2011; Bruckbauer and Cox 2021). Desiccation, UVC, and ionizing radiation cause similar damage mediated by reactive oxygen species (ROS) to vital physiological functions of cells within hours to days after exposure, resulting in death or long-lasting genetic consequences among survivors (Daly 2009).

The Rover Environmental Monitoring Station (REMS) onboard the Mars Science Laboratory (MSL) mission has a UV sensor (UVS) that has been measuring since 2012 the UVC radiation flux at the surface of Mars. As measured directly by REMS (Vicente-Retortillo et al. 2020), the flux of solar UVC at the surface of Mars depends on time of day and atmospheric, seasonal, and orbital circumstances, reaching a maximum of 2.5-3.5 W/m². Because of the thin atmosphere, most GCR protons impact the ground and generate additional ionizing radiation in the top 10 cm (Hassler et al. 2014). However, within mission-relevant time periods, the bactericidal effects of GCR are considered to be insignificant compared to the sterilizing power of UVC at the surface.

The following circumstances are known to dramatically affect an organism’s radiation and desiccation resistance:

• Ambient nutrient conditions. The presence of nutrients governs (i.e., enables) recovery (Venkateswaran et al. 2000; Ghosal et al. 2005).
• Temperature. Freezing increases radiation resistance (Richmond et al. 1999; Musilova et al. 2015).
• Hydration. Organisms are more tolerant of radiation when desiccated (NRC 2002a; Dartnell et al. 2010).

Representing distinct habitability settings, the dominant stressor likely to limit dormant life is radiation (Teodoro et al. 2018)—at the surface, UVC (200-300 nm); within the uppermost subsurface (<10 cm) GCR effects are amplified (Hassler et al. 2014); and in the deep subsurface (>10 m), internal background ionizing radiation. Considering these habitability settings, the committee discusses (1) the UVC radiation, ionizing radiation, and desiccation resistance characteristics of environmentally robust (extremotolerant) prokaryotes and simple eukaryotes; (2) the radiation and desiccation resistances of the

organisms corresponding to survivability over thousands of years shielded beneath the subsurface, but not in surface environments exposed to UVC; and finally (3), survivability in the subsurface.

Survival Characteristics of Extremotolerant Microorganisms

Many radiation- and desiccation-resistant organisms can recover from ionizing radiation doses that are equivalent to surviving millions of years of internal background radiation in the Martian subsurface (Box 3.1). Background ionizing radiation in the permanently frozen subsurface of Mars (>10 m) is likely to be the same as on Earth, ~0.5 mGy/yr. However, in the near-surface (~10 cm) of Mars, the background dose rate for GCR is ~250 times greater, ranging from 76 to 96 mGy/yr (Gy = J/kg = 100 rad) because of the lack of atmospheric shielding, and similar to the dose rate on the lunar surface (Hassler et al. 2014). In comparison, surface microorganisms, or those carried by the wind as aerosols and suspended in the Martian atmosphere, will be inactivated relatively quickly by UVC reaching 2.5-3.5 Wm⁻² (Vicente-Retortillo et al. 2020).

Microbial Survivability at the Martian Surface

Over the last three decades, numerous microorganisms have served as models considering the potential for survival and proliferation of life beyond Earth, as well as the ability of life to survive long periods of metabolic dormancy in high-radiation, frozen, and desiccated states (Holm-Hansen 1967; Slade and Radman 2011; Sharma et al. 2017). As most, if not all, of the characteristics required for survival of life exposed to radiation and desiccation are embodied by Deinococcus vegetative cells and Bacillus subtilis spores, the committee summarizes here the survival limits of D. radiodurans (Makarova et al. 2007) and B. subtilis, where the radiation dose needed to kill 90 percent of a population is the survival index D₁₀ (Dartnell et al. 2010):

• UVC: D. radiodurans D₁₀ in aqueous state: 2 kJm⁻² (Makarova et al. 2007); Bacillus subtilis spores D₁₀: 0.5 kJm⁻² (Schuerger 2015).
• Ionizing radiation: D. radiodurans D₁₀ frozen at −79°C: 50 kGy² (5 Mrad) (Richmond et al. 1999); B. subtilis spores D₁₀: 3 kGy (Granger 2011).
• Desiccation: likely decades Deinococcus cells and Bacillus spores (Daly 2009).

The UVC flux at the surface of Mars reaches 2.5-3.5 Wm⁻² (Vicente-Retortillo et al. 2020). This is sufficient to inactivate most radiation-resistant prokaryotes in a matter of hours to days, depending on the season; including Deinococcus phoenicis (1P10ME) isolated from the cleanroom floor at the Kennedy

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² Gray, defined as J/kg.

Space Center (Vaishampayan 2014). For this report, the committee adopts a surface survival time of 1-2 sols.

Microbial Survivability in the Martian Subsurface

A critical issue for the preservation of contaminating microorganisms in the Mars subsurface is ionizing radiation.³ If an organism similar to D. radiodurans were to be maintained as fully cryptobiotic, or simply frozen and desiccated in a subsurface environment of Mars, the theoretical accumulated dose maximally allowed without overwhelming an ecological population might approach 50 kGy. At the uppermost subsurface (<10 cm) of Mars, the background dose rates for all ionizing radiation range from 76 to 96 mGy/yr (Hassler et al. 2014). Therefore, the theoretical maximal survival dose of desiccated and frozen Deinococcus bacteria in Mars uppermost subsurface environments should be reached in ~0.5 Myr. Other microorganisms have been shown to effectively repair radiation-induced DNA damage under freezing conditions in the absence of net growth (Dieser 2013). Studies of ancient ice on Earth suggests that at temperatures below −20°C, microbial DNA is likely to degrade over a period of ~1.1 million years (Bidle et al. 2007). Therefore, any contaminating viable microorganisms that become frozen in the subsurface could remain dormant and viable for thousands of years, well beyond mission-relevant time frames, but not considered to be harmful contamination.

In summary, there is no evidence for a lower temperature limit for the existence or preservation of life on Earth, and freezing temperatures alone are not expected to affect the survival of microorganisms on Mars. Similarly, desiccation or GCR, alone or in combination with freezing, are also not likely to affect survival over periods of at least decades to millennia, depending on temperature and exposure to environmental oxidants. However, terrestrial microorganisms on the surface, or suspended in the atmosphere, of Mars will be largely unprotected from the deleterious effects of UVC radiation.

Transport of Biota

Long-distance movement of microorganisms is dependent on their interactions with physical processes (e.g., wind, micrometeoroid gardening, impact cratering, or possible brine channels), or transport by attachment to vehicles. Here, the committee considers atmospheric transport as a primary mechanism, with attachment to rovers or other devices as being secondary, because atmospheric transport would result in longer travel distances over shorter periods of time. Organisms attached to rovers or other devices may encounter some shielding from surface conditions that also could positively influence their survival. Future human exploration of Mars will increase the potential for microbial transport, and that issue is addressed in Chapter 5.

The key differences between the atmosphere of Mars and Earth that most impacted the committee’s deliberations on planetary protection are pressure and water content. The Martian atmosphere is not sufficiently dense to attenuate solar UVC radiation or provide protection to suspended Earth organisms. The dearth of atmospheric water greatly extends retention of dust in the atmosphere, giving rise to planet-wide dust storms (Aoki et al. 2019), which may transport organisms long distances.

On Earth, atmospheric transport is responsible for the intercontinental dispersal of microorganisms (Shivaji et al. 2006), with dust-associated cells in particular transiting thousands of kilometers (Stres et al. 2013; Liu et al. 2016; Santibáñez et al. 2018). Indeed, viable bacteria have been isolated from ice deposits on Earth that exceed 100,000 years in age. These microorganisms may be shielded by particle attachment, be transported in spore form, or deploy other defense mechanisms such as the production of extracellular

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³ For the purposes of this report, unless explicitly defined otherwise, the committee adopts the following definitions: uppermost subsurface as the upper 10 cm, shallow subsurface as the upper 1 m, and deep subsurface as deeper than 10 m.

polysaccharides and pigments that protect cells from stresses such as UV and desiccation, although the detection of individual bacterial cells in ice cores (Santibáñez et al. 2018) suggests that non-spore-forming and non-encased cells can also remain intact during atmospheric transport (Shivaji et al. 2006). Together, these pieces of evidence suggest that microbial cells, both with and without obvious defense mechanisms, survive atmospheric transport on Earth.

In considering atmospheric transport as a microbial dispersal mechanism on the Martian surface, the committee discussed:

• The potential source reservoir
• The transport mechanisms and challenges associated with transport
• The potential sinks and conditions therein

The Potential Source Reservoir

Forward-contaminating microorganisms that are transported must first be delivered by spacecraft, and the total biomass is limited by the initial level of contamination and loss of microbial cells during transit to Mars. For spacecraft, culture-based estimates of microbial burden are dependent on colony formation on nutrient agar plates. However, such growth assays are blind to innumerable terrestrial microorganisms that are unculturable yet viable (Rappé and Giovannoni 2003). Upon landing, surface abrasion by dust and wind is inevitable on landers and rovers, thereby mobilizing residual surface microorganisms. Therefore, time aloft in the wind is relevant to define the planetary protection level for a given landing site, based on its proximity to potential habitable environments (p. 33).

Mechanisms of Transport and Associated Challenges

Wind transport trajectories and distances will depend principally on prevailing lower and upper atmosphere conditions, turbulence, dust sedimentation rates, and time of day. Notwithstanding the low atmospheric pressure, the prevailing winds can reach 100 km/h and transport fine sand grains (Toon et al. 1977), which can remain suspended in the atmosphere for months to years (Moores et al. 2016); in net, however, the dynamic pressure of even these strong winds is significantly lower than on Earth due to Mars’ lower atmospheric pressure. Sedimentation rates of lofted dust on Mars are greatly reduced, relative to Earth, by the lack of precipitation and low atmospheric water content. During transport, cells will be exposed to UVC and GCR radiation, low temperature, and low humidity, requiring that cells deploy protective mechanisms or travel attached to shielding particles in order to survive.

Meteorite impact ejecta can mobilize encased dormant microorganisms into the atmosphere, although those near the impact point will be inactivated by the heat generated by impact. Microorganisms mobilized into the atmosphere would most likely be inactivated within a matter of hours to days by the high UVC doses received during daytime atmospheric transport. Microorganisms aerosolized from the surface by dust devils, helicopters, thrusters, impacts, and avalanches, or carried by the wind, are expected to be inactivated within 1-2 sols by UVC (p. 19).

The Potential Sinks and Conditions Therein

The survival of Earth-sourced organisms delivered to the surface of Mars and transported by winds or other disturbances will depend upon their delivery to a suitable environment that can support life. The presence of water and protection from UVC and GCR will increase the chances of survival. Potential habitats include brine channels in ice, subsurface aquifers with a surface expression, subglacial ice melts, caves, and endolithic environments containing hydrated minerals. Upon arrival at one of these favorable

environments, an adequate supply of nutrients and energy sources would be needed to sustain life. While it is established that cultivable radiation-resistant prokaryotes and fungi require nutrient-replete conditions for recovery from radiation, there is evidence from extremely dry, nutrient-poor environments on Earth that life can be sustained in the presence of very minimal nutrients, but the minimum required for growth is not known (Goordial et al. 2016).

Proliferation of Biota in Subsurface Habitats

Though understanding of Martian surface conditions as discussed here is fairly well established based on spacecraft data from the last several decades, a critical piece of information that is still lacking is adequate knowledge of subsurface access points, such as cave entrances. Current knowledge of candidate cave openings (Cushing 2015) is based primarily on High Resolution Imaging Science Experiment (HiRISE) and MRO Context Camera (CTX) images. HiRISE imagery only exists for 2-3 percent of the Mars surface. The resolution of CTX allows for identification of candidate cave openings as small as 25 m across. Until high-resolution images of the Mars surface are acquired that allow for the identification of cave openings as small as ~1 m, caution must be exercised in planetary protection measures. Proliferation of microbial life introduced from Earth is a major concern for planetary protection. Below, the committee discusses potential impacts of the Martian environment on proliferation in the Martian subsurface.

Temperatures Required for Growth

Microbial growth and cell division of cultivated microorganisms have been reported at temperatures as low as −15°C (258 K, bacteria [Mykytczuk et al. 2013]) and −18°C (255 K, yeast [Collins and Buick 1989]). Growth and metabolic activity were deemed probable at temperatures down to at least −20°C (253 K) in permafrost [Rivkina et al. 2000]). These temperatures are substantially warmer than the proposed limits for metabolic activity and growth (e.g., −40°C or 233 K [Price and Sowers 2004]) and warmer than temperatures at which metabolic activity, which may not lead to growth, has been detected (−25°C or 248 K [Mykytczuk et al. 2013]); −33°C or 240 K [Bakermans and Skidmore 2011]). Based on current understanding of microbial growth, microorganisms deposited in subsurface areas on Mars at temperatures below ~ −20°C (253 K) are not likely to grow. The committee adopts the −28°C (245 K) limit, which includes a safety margin to the observed temperature of −18°C (255 K) for cell division, as proposed for Mars Special Regions (Kminek et al. 2015; COSPAR 2020) by COSPAR.

Liquid Water

The range of conditions under which water occurs in a liquid state and is available is expanded by the addition of solutes to the solution, which decreases the water activity from that of pure water, which has water activity of 1. The experimentally determined water activity limits for the growth of fungi and bacteria are 0.605(van der Wielen et al. 2005) and 0.635, respectively, and the lower limit for bacteria and archaea is proposed to be 0.611 (Stevenson et al. 2014). The committee adopts the water activity value of 0.5 used by COSPAR for terrestrial microorganism replication (COSPAR 2020).

Nutrient Conditions

Terrestrial extremotolerant organisms, which have been cultured and are thus available for study, require a source of macronutrients for recovery from radiation and desiccation; therefore, organisms delivered to subsurface environments in either desiccated or radiation-compromised states would likely

share this requirement. While such nutrients are rare or absent from Martian surface environments, there is currently insufficient information to determine their availability in the subsurface.

After considering all the conditions and locations for relaxed bioburden requirements discussed above, the committee reached the following conclusion:

SPECIAL REGIONS

In planetary protection policy, regions on Mars where terrestrial organisms are likely to replicate, or that have a high potential for the existence of extant Martian life, are called “Special Regions.” The COSPAR Planetary Protection Policy (COSPAR 2020) provides the most recent listing of Special Regions on Mars. No Special Regions have been identified on the basis of possible extant Martian life, but the COSPAR list of the regions or terrain types treated as Special Regions until more is known follows:

• Observed Features
  • Gullies and associated bright streaks
  • Subsurface cavities
  • Subsurface below 5 m
  • Recurring slope lineae (RSL) (confirmed and partially confirmed)
• Features Not Yet Observed
  • Groundwater
  • Sources of methane
  • Geothermal activity
  • Modern outflow channels
• Features That Require Case-by-Case Evaluation
  • Pasted-on terrains
  • Dark streaks
  • RSL (candidate)

The characterization as potential Special Region is based on the interpretation of data from remote sensing instruments, namely imaging, imaging spectroscopy, gamma ray and neutron spectroscopy, and radar. Approximately 3 percent of the Martian surface has been imaged at the highest resolution of MRO HiRISE. In combination with data from CRISM and observations by the stereo-imaging system Cassis on ExoMars TGO, better characterization of features is expected in the near future and will aid in confirmation of their status as Special Regions. A big information gap exists for the subsurface region; gamma ray and neutron spectroscopy provide information on the top centimeters down to 1 m, however, a mission such as the proposed ICE Mapper could provide characterization of the top 10 cm of the surface. Neither MarsExpress MARSIS nor MRO SHARAD have the required vertical resolution to provide information on any shallow water at low latitudes.

Recurring Slope Lineae and Gullies

In the 2014 MEPAG SR-SAG2 report (Rummel et al. 2014), some types of gullies and recurring slope lineae (RSL) were considered “Uncertain but treated as Special.” (Here the MEPAG report authors used the term “Uncertain” to refer to environments for which proof of capability of supporting microbial growth is not possible to obtain, but which simultaneously demonstrate the temperature and water availability conditions to allow propagation.) RSL are narrow (0.5-5 m), low-albedo features that lengthen over time down steep slopes during warm seasons; they appear and disappear seasonally. At the time of the SR-SAG2 meeting, MRO HiRISE had detected RSL, which were considered to be possibly related to the presence of water. However, the preponderance of recent scientific results that the committee heard supports the position that RSL and gullies are formed by dry processes.

Following the SR-SAG2 meeting, researchers have pointed out problems with liquid water interpretations of RSL formation. Though some CRISM results seem to indicate hydration features associated with RSL (Ojha et al. 2015), further work cited data processing artifacts in CRISM measurements at 1.9 and 2.1 mm, where hydrated perchlorates exhibit absorption features (Leask et al. 2018). Melted ice and/or brines have been suggested as a source of the RSL; however, many RSL are observed on equator-facing slopes at mid-latitudes and in equatorial canyons, such as Valles Marineris, and are not expected to accumulate enough ice for this to occur (Stillman et al. 2020). Similarly, aquifers have been suggested as the source of RSL (Abotalib and Heggy 2019), which is difficult to explain without having any evidence of aquifers. Also difficult to explain is how the groundwater would access the relatively high-elevation origination points of the RSL. Furthermore, as discussed by Wray (2020), several researchers have estimated the abundance of water required to explain RSL. Volume estimates range from several to 10⁶ cubic meters of water, which are challenging abundances to derive at the surface of Mars. More recent evidence points to flows of dry sand and dust as explaining RSL (Dundas et al. 2017). From topographic analysis, these research groups conclude that the terminal slopes of RSL match the stopping angle for granular flow of cohesionless sand in active Martian aeolian dunes. RSL observed in Eos Chasma are also inconsistent with models for water sources.

Similarly, gully morphologies on Mars can be explained by processes not involving liquid water. Early studies interpreted gullies as evidence for recent, and perhaps current, flowing water. The temporal coverage of MRO HiRISE has been utilized to show that gullies demonstrate a clear seasonality and are strongly correlated with the occurrence of CO₂ frost (including spectral evidence of CO₂), making the conditions too cold to be related to liquid water. “Linear” dune gullies are also seen to be active, with present-day changes apparently driven by sliding blocks of CO₂ frost (Diniega et al. 2013). Present-day changes in gullies suggest that non-aqueous processes are major contributors to gully formation and suggest that gullies form entirely by CO₂ frost processes.

Subsurface Cavities

The 2019 National Academies of Sciences, Engineering, and Medicine Astrobiology Strategy report recommended emphasis on research and exploration of subsurface environments based on the astrobiological potential of extraterrestrial caves. Martian caves, particularly lava tubes, are of astrobiological interest (Carrieret al. 2020) and are considered potential Special Regions due to the protection they provide from surface radiation and temperature fluctuations. The Mars Global Cave Candidate (MGC3) catalogue (Cushing 2017) lists more than 1,200 candidate cave entrances (Figure 3.1), mostly in the volcanic Tharsis and Elysium regions, based on images from CTX and HiRISE cameras. CTX images have resolution of ~6 m per pixel, which is sufficient to identify cave entrance candidates as small as ~25 m across. These volcanic caves are expected to have interconnected fracture networks (Bouley et al. 2018). Because most known or postulated caves are at relatively high elevations on Mars, future missions using current entry, descent and landing techniques will likely not choose landing sites in the vicinity of cave entrances (Titus et al. 2021).

Martian caves remain largely uncharacterized, but are thought to be one to three times more voluminous than Earth caves (Sauro et al. 2020). While thermal conditions within caves are unknown, a cave roof thickness greater than 1 to 2 m reduces the amplitude of the ground temperature variation during the day. Cave roof thickness also has a significant impact on the air temperature in the cave, resulting in negligible influence of the surface temperature on the environment inside the cave. Within the caves, temperatures are likely cold (<−25°C or 248 K) and thus could harbor metastable water ice (Williams et al. 2010). Cave locations and predicted ice accumulation rates (Kearney et al. 2021) have been mapped. It is unknown whether terrestrial microbial life could proliferate in Martian caves, though researchers determined that ice in terrestrial caves can contain metabolically active bacteria (Carrier et al. 2020).

Global Subsurface; Groundwater

The deep subsurface of Mars is of astrobiological interest based on terrestrial analogues (Carrier et al. 2020). Mars was characterized by an active hydrosphere early in its history (Carr and Head 2010); significant quantities of water formerly on the surface may have retreated to subsurface aquifers beneath an ice-rich permafrost zone (Clifford and Parker 2001; Figure 3.2). Multiple sedimentary deposits on the Martian surface have been attributed to upwelling groundwater in the ancient past (Salese et al. 2019), providing evidence of subsurface aquifers earlier in Martian history. Rummel et al. (2014) noted that groundwater (at any depth) would be considered a special region on Mars if found to exist. On Earth, groundwater and redox energy from radiolysis, namely α, β, and γ radiation from decay of radionuclides, can support life. Microorganisms in Earth’s subsurface have been found to be actively metabolizing in the

presence of groundwater after 10⁶-10⁹ years of being isolated from the surface powered by radiolysis, which is insufficient to inactivate microbial communities (Tarnas et al. 2021) but sufficient to produce H₂ and sulfates to sustain sulfate-reducing bacteria wherever groundwater exists (Sauvage et al. 2021). Indeed, Tarnas et al. (2021) show that the source regions of the Martian meteorites (SNC and regolith breccia) would all produce sufficient redox energy to support sulfate-reducing life where groundwater is present.

Whether groundwater aquifers persist to this day on Mars is unknown. Pure water aquifers, if they exist, should be several km deep (Clifford et al. 2010) (deeper than the few hundred meter detection range of SHARAD or MARSIS). Briny aquifers could be shallower, but there is no evidence of these from radar at depths to ~200 m (though SHARAD has blind spot 0-10 m). Carrier et al. (2020) point out that the D/H ratio supports little water loss since the Hesperian (roughly 3000 Mya), and thus supports the existence of widespread liquid groundwater. However, much of Mars’ ancient water may be bound in hydrated minerals in the Martian crust (Scheller et al. 2021).

At the Insight landing site, Manga and Wright (2021) used the seismic shear wave velocity (Vs) to demonstrate the local absence of an ice-saturated cryosphere in the upper 8-11 km of the Martian crust. The lack of such a low latitude cryosphere implies that an underlying aquifer could be depleted (Grimm et al. 2017), which has implications for aquifers elsewhere, unless Mars aquifers are compartmentalized or unconfined by an overlying cryosphere (Manga and Wright 2021).

Geothermal Activity, Sources of Methane, and Sites of Recent Seismic Activity

Methane breaks up in the presence of ultraviolet solar radiation. Based on photochemical models and current understanding of the composition of the Martian atmosphere, methane has a chemical lifetime of about 300-600 years, which is very short relative to geological time scales. This implies that the methane that is observed today (Formisano et al. 2004; Mumma et al. 2009; Webster et al. 2021) cannot have been produced 4.5 billion years ago, when the planets formed. There are several alternatives that could explain its presence on Mars.

1. Geological origin. The methane could be produced, for example, by the oxidation of iron, similar to what occurs in terrestrial hot springs, or in active volcanoes. This gas could have been trapped in solid forms of water, or “cages,” which can preserve methane of ancient origin for a long time. These structures are known as “clathrate hydrates.”
2. Geochemical origin. Serpentinization could also produce the abiotic methane. Serpentinization is a geological low-temperature metamorphic process involving heat, water, and changes in pressure. It occurs when olivine, a mineral present on Mars, reacts with water, forming serpentine; in the presence of carbon dioxide, the hydrogen can combine with the carbon to form methane. On Mars it is possible to find all the primary ingredients of olivine and carbon dioxide, but the chemical reaction needs liquid water to occur. This implies that, if the Martian methane comes from serpentinization, it could be related to subsurface hydrothermal activity.
3. Biological origin. The discovery of microbial life 2 to 3 km beneath the surface of the Witwatersrand basin in South Africa (Ward et al. 2004) led scientists to consider that similar organisms could live, or have lived in the past, below the permafrost layer on Mars. By analogy with Earth, the biological origin of Martian methane could be explained by the existence of microorganisms, called methanogens, existing deep under the surface, and producing methane as a result of their metabolism. If the origin is biotic, there are two possible scenarios: either long-extinct microorganisms, which disappeared millions of years ago, have left the methane frozen in the Martian subsurface, and this gas is being released into the atmosphere today as temperatures and pressure near the surface change, or some very resistant methane-producing organisms still survive.

Concentrations of methane were observed in 2003 and 2006 in three specific regions of Mars—Terra Sabae, Nili Fossae, and Syrtis Major (Formisano et al. 2004; Mumma et al. 2009; Yung et al. 2018; Webster et al. 2021). Deep liquid water areas below the ice layer would be able to provide a habitat for microorganisms, or a favorable place for the hydro-geochemical production of methane. Further processing in the Martian atmosphere may play an important role that accounts for the observed seasonal variability. Recent combined results from Curiosity and ExoMars TGO (Formisano et al. 2004; Mumma et al. 2009; Webster et al. 2021) show a day-to-night variability, with significantly greater amounts of methane being observed at night. This suggests that methane accumulates near the surface at night, but is dispersed through the atmosphere and drops below detection limits during the day. Whether geochemical or biochemical in origin, the measured variation in concentrations of methane indicates that Mars could still be active today.

PLANETARY PROTECTION IMPLICATIONS: CONDITIONS AND LOCATIONS FOR RELAXED BIOBURDEN REQUIREMENTS

Mars Conditions Where Limits for Microbial Growth Are Met

Modelled Subsurface Temperatures

The committee adopts a lower temperature limit for microbial growth of 245 K (−28°C) (cf., discussion, p. 26). The depth at which maximum annual temperatures fall below a given value, for any given location, is difficult to constrain with available data, and depends on latitude, thermal conductivity, bulk density, heat capacity, and vertical heterogeneity of the subsurface. Given those caveats, the

committee provides rough constraints on temperature versus depth below using model-based estimates of mean annual and daily temperatures (Kieffer 2013).

Estimates of mean annual temperature at the surface are a reasonable approximation for subsurface temperature at ~three seasonal skin depths,⁴ which for regolith corresponds to a depth of ~2 m (Table 3.2).⁵Kieffer (2013) modeled mean annual surface temperatures using Mars Global Surveyor TES-derived thermal inertia values (Putzig and Mellon 2007; Figure 3.3). The resultant map illustrates that temperatures at ~2 m depth are <230 K (−43°C) for much of the planet. However, it is important to note that these modeled temperatures assume homogeneous surface materials under each pixel, with thermal inertia values corresponding to those estimated from the upper few centemeters of the surface. Subsurface temperatures for buried water ice would be warmer than these modeled values because of its higher heat capacity and thermal conductivity than soil. Better constraints on material properties with depth and crustal heat flow are needed to improve estimates of subsurface temperatures. A variety of orbiter- and lander-based observations such as improved diurnal and seasonal temperature coverage, imaging radar, and in situ porosity, thermal conductivity, and crustal heat flow measurements would provide such constraints.

A similar approach can be used to assess maximum temperatures that might occur at depths shallower than 2 m. Mean daily temperatures approximate the subsurface temperature on that day at approximately three diurnal skin depths, which ranges between ~10-60 cm depending on the thermal inertia of the material (Putzig and Mellon 2007; Table 3.2). The committee used the KRC thermal model from Kieffer (2013) to model mean daily temperatures on the warmest sol of the year, as a function of latitude, for different material types representative of regolith and potential shallow subsurface materials (Figure 3.4).⁶ This analysis suggests that maximum subsurface temperatures fall well below 245 K (−28°C) somewhere in the upper ~10-60 cm of the surface at northern latitudes. In the southern hemisphere, temperatures >245 K extend to depths of at least ~60 cm depending on the material (Figure 3.4).

Temperatures exceeding 245 K (−28°C) are also expected in the deeper subsurface (below tens of meters to >100 m, depending on average surface temperature), from geothermal heat.⁷ All of these depths are estimates and could vary with multiple factors that were unaccounted for in this analysis (e.g., topographic variability with longitude, layered subsurface properties, heat flow values different than assumed).

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⁴ The mean surface temperature over a diurnal or annual period should be a close approximation of the temperature at ~three times the thermal diurnal or seasonal skin depth (e.g., where the amplitude is reduced to ~5 percent), respectively, assuming homogeneous material properties with depth and not accounting for crustal heat flow.

⁵ Values are calculated from Equation 3 of Mellon and Jakosky (1993) using thermal inertia, density, and heat capacity values from Mellon et al. (2004). Using Equation 3 from Mellon and Jakosky (1993), values of heat capacity, density and thermal inertia from Mellon et al. (2004) and Putzig and Mellon (2007). Each of the example materials in Table 3.2 exhibit a range of thermal inertia, density, and heat capacity values; for example, thermal inertia values of sands can range from ~150-350 J m-2 K-1 s-1/2 depending on grain size (e.g., Presley and Christensen 1997), and rock can range from ~600-2500 J m-2 K-1 s-1/2, depending on porosity, composition, etc. (e.g., Kahle 1980; Fergason et al. 2006).

⁶ The committee used KRC (Kieffer 2013), a publicly available thermal model (http://krc.mars.asu.edu) that has been used in a range of studies of Martian temperatures and thermophysical properties (e.g., Fergason et al. 2006; Hamilton et al. 2014; Vasavada et al. 2017; Piqueux et al. 2019, 2021). Version 3.6.5. was used, after Piqueux et al. (2021, https://doi.org/10.1029/2021JE006859), through the Davinci software interface (available from http://davinci.asu.edu). Longitude was set to 0°, and albedo was set to 0.16; all other parameters were set to the model default values. The model retrieves elevation from a MOLA-derived gridded elevation map based on user-defined latitude and longitude.

⁷ Assuming a crustal heat flow of 20 mW m^-2 (Siegler et al. 2017) and thermal conductivity of ~0.04 W m^–1 K^–1 (Grott et al. 2021) the thermal gradient would be ~0.5 K/m (Grott et al. 2021). Average surface temperatures of 160-230 K were used (Figure 3.3).

TABLE 3.2 Example Geologic Material and their Thermophysical Properties

Water Activity

The committee adopted a water activity limit of 0.5 (cf., p. 26). Liquids with water activity >0.5 could form where salts are in contact with ice, contained within ice, or via deliquescence at a range of temperatures at least down to 198 K (or −75°C) (Rivera-Valentín et al. 2020). Such conditions could be met in the subsurface at a range of depths; however, to be stable against evaporation, temperatures must remain below ~205 K (or −68°C) (Rivera-Valentín et al. 2020). In the upper ~15 cm of the subsurface, the combination of low temperatures and high relative humidities necessary for brine formation are met for up to six consecutive hours per night over portions of the Martian year at high latitudes, leading to a

total duration of brine stability of up to 2 percent of the Martian year at the highest latitude modeled (60°N) (Rivera-Valentín et al. 2020). Under closed conditions (i.e., where there is no atmospheric exchange), however, the temperature limit for brine stability against evaporation would be removed and the 245 K (or −28°C) temperature limit for growth could be met in portions of the shallow subsurface.

Thus the committee concludes that temperature and water activity conditions that allow subsurface microbial growth (as adopted in this report, namely, T >245 K (−28°C) and a_w >0.5) are possible for portions of the upper few tens of cm of the Martian subsurface in closed-system environments (meaning, no atmospheric exchange) within ice or regolith, based on current knowledge of Mars. Other locations that meet the criteria for growth include the deeper Martian subsurface (>tens of m) or any hydro/geothermal heating environments that might be discovered.

Deliquescence could potentially occur in the shallow subsurface, but there is still limited knowledge about the kinetics. Deliquescence might be a slow enough process such that temperature cycling may inhibit it from occurring (Fischer et al. 2014; Rivera-Valentín et al. 2020). However, brine formation through salt-ice interactions occurs on much faster time scales (Fischer et al. 2016). Additionally, known abundances of deliquescent salts, from Phoenix and Mars Science Laboratory measurements, constitute only ~0.5 wt% of the Martian regolith or less (Hecht et al. 2009; Kounaves et al. 2010; Leshin et al. 2013), suggesting that such brines would be dispersed and forming at the grain scale. In terms of both H₂O volume and timescale of brine formation, subsurface ice might provide a more likely location for potential growth. The state of knowledge on Martian subsurface ice distribution is described below.

Subsurface Ice Distribution

Existing radar, neutron spectroscopy, thermal and high-resolution imaging data sets provide incomplete knowledge on where permafrost and buried ice sheets exist, with each data set having its own set of limitations in vertical and spatial resolution. Sounding radar from SHARAD and MARSIS have vertical resolutions of ~10 and 100 m, respectively, permitting resolution of the base of ice sheets (Bramson et al. 2015) and other ice deposits within lobate debris aprons (Plaut et al. 2009). Buried ice sheets (“excess ice,” which exceeds the regolith pore space) up to 80-170 m thick have been observed at latitudes as low as 38°N (Bramson et al. 2015; Stuurman et al. 2016); because the top of the ice sheets are not resolved, they must be within the top ~10 m of the subsurface (Plaut et al. 2009). Recent impacts that expose nearly pure ice show that excess ice exists within the upper meter of the surface at latitudes as low as ~39°N (Dundas et al. 2014).

Though the distribution of recent impact-excavated ice provides visual confirmation of ice in the upper ~1 m, it does not provide complete spatial coverage of the mid- to high-latitudes due to the point-sampling nature of this data set. Neutron spectroscopy and infrared imagers (Figure 3.5) provide a means to detect ice within the upper ~meter. Neutron spectroscopy (~500 km/pixel) indicates that ground ice is present within the upper meter across much of the high latitudes, poleward of ~60°S and ~45°N (Boynton et al. 2002). Thermal measurements at coarse spatial resolution (~3 pixels per degree) suggest that near-surface excess or pore-filling ice is nearly continuously present⁸ in some regions at latitudes poleward of 35°N and 60°S (Piqueux et al. 2019). Note, however, that neutron and infrared measurements are not sensitive to regolith properties or ice presence below ~1 m depth.

It is important to note that thermal-based studies cannot effectively assess depth to ice below 10 cm in the equatorial latitudes (gray regions in Figure 3.5a) due to the lack of seasonal variability in surface temperatures to the equator and southern hemisphere spring corresponding with high dust loadings (Piqueux et al. 2019). Thus, lack of detection in thermal-based studies does not indicate an absence of shallow ice at low latitudes. In fact, there is other evidence that shallow water ice may exist at equatorial latitudes. In the southern hemisphere, Vincendon et al. (2010) noted a lack of detection of CO₂ frosts on pole-facing slopes poleward of 25°S. These slopes should be cold enough to condense CO₂ frosts if composed only of regolith; Vincendon et al. show that a high thermal inertia material (water ice) in the subsurface must be present in order to prevent the surface from cooling below the CO₂ frost point. This line of reasoning assumes that there are no other potential reasons for lack of spectral detection of CO₂ frost, and it does not provide assessment of how continuous such a water ice-bearing layer might be at these latitudes. Excess hydrogen is also measured from the Mars Odyssey neutron spectrometer in some equatorial regions and could plausibly be due to buried ice (Feldman et al. 2011; Wilson et al. 2018).

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⁸ The thermal-based map in Figure 3.5 is averaged over 3 ppd scales. Depth to ice is likely to be highly spatially variable at “cm to regional” scales (Piqueux et al. 2018), and there is evidence for this based on terrestrial observations, high-resolution thermal imaging observations from THEMIS, Phoenix observations, and theoretical modeling.

Models of ground ice stability predict that ground ice should not be stable in the upper meter of regolith at latitudes <49° (for flat ground) or <25° (for pole-facing slopes) in the current climate (Mellon and Jakosky 1995; Schorghofer and Aharonson 2005; Aharonson and Schorghofer 2006). However, these models assume diffusive equilibrium with the atmosphere. Subsurface conditions (e.g., duricrusts, salts) that prevent diffusive exchange with the atmosphere could potentially permit metastability of ancient ice

at lower latitudes and shallower depths than model predictions for current climate (Feldman et al. 2011). Indeed, many shallow water ice detections have been reported to be outside the range of predicted stability (Piqueux et al. 2019; Morgan et al. 2021) and some have been suggested at equatorial latitudes⁹ (e.g., in the Medusa Fossae Formation [Feldman et al. 2011; Wilson et al. 2018]).

In summary, current observations are consistent with the presence of water ice in much of the Martian shallow subsurface (<1 m depth) at latitudes poleward of 35°N and ~50°S, and there is some evidence of water ice at equatorial latitudes. There are no available observations to confidently rule out ice presence between 1-10 m depth at any latitude, or between ~10-100 cm depth at equatorial latitudes between ~35°N and ~50°S. There are locations where model results suggest ice may be absent in the upper ~0.5 m (green to red in Figure 3.5a), but higher spatial resolution temperature observations would be needed to refine modeled ice depth at mission-relevant spatial scales. Overlapping temperature observations at 100 m/pixel from the Mars Odyssey THEMIS instrument are available across much of the mid-latitudes spanning multiple seasons, however this resolution is likely not sufficient to rule out ice presence at mission-relevant scales. In short, remote observations are excellent means for detecting water ice, but not for confirming the absence of such ice.

Potential Locations for Relaxed Bioburden Requirements

Given the conditions described above, the committee assesses some potential regions on Mars where bioburden requirements for future missions might be relaxed. Knowledge about the Martian subsurface is incomplete, which poses challenges for finding locations that will avoid conditions for growth with certainty (p. 33). As described above, there could be closed-system environments at low latitudes where ice or brines could be stable or closed-system environments in ice where brines could be stable. Other examples might include possible hydro/geothermal environments or void spaces within shallow bedrock.

Regardless of the uncertainties, some of these environments are likely to have limited connectivity, and thus some contamination and growth could potentially be acceptable because it would be contained and thus would not constitute harmful contamination. Replication may be acceptable within a contained region, as long as proliferation outside that region does not occur:

1. Regions where no ice is detected on the basis of neutron and thermal data analysis (Figure 3.5 and Figure 3.6). Though closed-system ice or brine could persist in these areas in the top 1 m, it is likely to be in low abundance and patchy distribution.¹⁰ Water activity conditions for growth would cease to exist outside each “patch.”
2. The upper few (~5-20) cm of the surface. Though conditions needed for replication could be present in the upper tens of cm in some locations, these would have to be closed-system environments. Such environments, if they exist, are likely to be discontinuous, at least in the upper few (~5-20) cm.¹¹ The upper surface of polar ice may also be acceptable for lower bioburden requirements; though water ice is exposed at the surface of the southern polar cap, maximum temperatures there (~200 K or −73°C [Titus et al. 2003]) are well below the limits for

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⁹ Neutron observations of shallow equatorial hydrogen are consistent with ice but also could be consistent with “significant abundances of hydrated minerals” (Feldman et al. 2011).

¹⁰ Higher abundances would be detected in these data sets; at equatorial latitudes ground ice is not predicted to be stable and would require a continuous diffusive barrier for which no evidence has been observed.

¹¹ At the Phoenix landing site (68.2°N 125.7°W), trench ice concentration was highly variable (Smith et al. 2009); trench depth ranged from 5-18 cm. Shallow subsurface salts that might form duricrust diffusive barriers were observed in discrete locations in Gusev crater (Spirit landing site, 14.5°S, 175.4°E) wheel trenches (~6-11 cm depth) (e.g., Wang et al. 2008) rather than as a continuous layer. From orbit, THEMIS-based observations suggest depth to ice is highly variable at the ~100 m scale (Bandfield 2007; Piqueux et al. 2018).

1. growth. Finally, the uppermost Martian subsurface is more subject to ionizing radiation (p. 19) that would further hinder growth.

To summarize the points discussed above, environments that permit microbial growth could be present in the top tens of centimeters of the shallow subsurface and below ~tens of meters in the deeper subsurface. However, such environments are likely discontinuous in (1) the upper few (~5-20) centimeters of the surface anywhere, and (2) the upper meter, in regions where no ice is detected from neutron or thermal observations. As such, these regions could be suitable for relaxed bioburden requirements. Exceptions to these guidelines for relaxed bioburden requirements would be areas in the vicinity of subsurface access points, such as an appropriately defined buffer zone (e.g., p. 42).

Permafrost, Subsurface Ice Sheets, and Polar Ice

Permafrost and buried ice sheets in the northern mid-latitudes and polar ice, which can be relatively continuous over km scales (Bramson et al. 2017), pose a challenge for assessing the likelihood of proliferation via subsurface mission activities because there is not sufficient information or detailed analyses needed to permit estimates of connectivity within the ice. Ice regions on Mars are of interest as potentially habitable zones (Carrier et al. 2020). There is risk of both lateral and vertical transport of biota via brine veins, channels, and fracture networks, which are common in terrestrial sea ice and permafrost (Lindensmith et al. 2016; Lieb-Lappen et al. 2017; Lieblappen et al. 2018). There is little information on mid-latitude or polar ice structure, porosity, or salt content that would allow for estimation of lateral extent and connectivity of these features. Furthermore, connectivity is likely spatially variable and thus universally applicable conclusions cannot be drawn about these subsurface ice environments.

The committee considered whether melting of the regolith is possible. As discussed, current neutron and thermal data do not detect subsurface water ice in the top 1 m of the Martian subsurface over much of the low latitudes, equatorward of roughly 30°-40° (Figure 3.6). Because of instrument resolution and sensitivity, the presence of water ice in these regions is possible, but because it is below detection levels, if water ice is present, it is likely patchy. Thus for landing sites in these regions, if melting by a radioisotope thermoelectric generator (RTG) occurred, that melt would remain localized and contained and thus would not represent a significant risk of harmful contamination. This conclusion is consistent with the findings of the 2019 Joint Workshop Report on Induced Special Regions (ISRs) (Meyer et al. 2019), which noted that ISRs could offer environments that would enable microbial replication but were unlikely to “globally contaminate Mars.”

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¹² Because ice is not detectable between ~1-10 m with existing thermal, neutron, or sounding radar subsurface reflection data, depths below 1 m could potentially harbor more continuous ice. The same connectivity concerns described below for permafrost and ice sheets (p. 40) apply to any putative ice environments below 1 m, hence the depth limit in this finding is restricted to 1 m. (The committee acknowledges that sounding radar surface returns can be used to search for low-density materials consistent with ice within the upper 5 m [Morgan et al. 2021]. However, uncertainty associated with determining the exact depth within the upper 5 m that any ice-like returns arise from, as well as the apparent spatial scatter in some of the data, presently introduces complexity and uncertainty in using the existing radar surface return data sets as a means to locate ice-free regions at depths below 1 m.) Although temperatures at depths below 1 m and down to several tens of meters are likely too cold for growth, uncertainty about habitat connectivity in notional ice raises concerns about the potential for transport to a more favorable location where growth and subsequent proliferation could occur (Morgan et al. 2021).

Subsurface Access Points and Sites of Astrobiological Interest

Locating Subsurface Access Points and Sites of Astrobiological Interest

There are numerous sites of interest that likely have not yet been contaminated by prior missions and that may be ideal near-surface targets to search for biosignatures of past life; as such, these sites need to be preserved for future astrobiological investigations and thus reduced bioburden requirements are not appropriate for these sites. Such regions include caves, hydrothermal mineral deposits, evaporite mineral deposits, phyllosilicate- or hydrated silica-bearing terrains, deltaic, alluvial or lacustrine deposits, and concentrated iron oxide deposits. These are identified by geomorphological analyses and spectral analyses (Carr 2007; Bell 2008).

Additional regions of astrobiological interest that should be avoided (for missions with relaxed bioburden requirements) include impact- or scarp-exposed water ice (Dundas et al. 2014, 2018) or fresh craters that do not expose water ice but that have formed within the last ~20 years (based on HiRISE). Several craters have been detected with data from multiple orbital passes and accompanying imagery (e.g., MRO HiRISE¹³), and may pose locations where Martian extant life might be detected (e.g., potential Special Regions [Rummel et al. 2014]).

The committee notes that knowledge of regions to be avoided (with relaxed bioburden), such as subsurface access points, is limited. MRO HiRISE maps the surface at ~0.3 or ~0.6 m/pixel scale, and has covered ~5.0 percent of the Martian surface (not counting repeat and stereo coverage of some sites). Nearly 100 percent of the surface has been mapped at ~6 m/pixel by the Context Camera on MRO.¹⁴ Thus, for the majority of the Martian surface, features of a scale smaller than 6 m have likely not been detected.

Wind Dispersal to Subsurface Access Points and Regions of Astrobiological Interest

Cave openings and regions of astrobiological interest (p. 28) constitute exceptions to a relaxed bioburden requirement. Even if a mission does not land on one of these sites, the risk of harmful contamination to these sites by wind transport needs to be assessed, and an appropriate buffer radius to reduce that risk needs to be determined. An appropriate buffer zone around the landing ellipse could be defined based on estimated bioload, modeled wind dispersal of particles, and maximum duration under which terrestrial biota could survive UV exposure under Martian conditions, such that probability of concentrated spore delivery to a region of astrobiological interest is reduced to an acceptable risk level. Alternatively, if a planned landing site is located near a site of astrobiological interest, the same approach could be used to establish bioburden limits to reduce risk to an acceptable level.

Here the committee provides an example of such an approach, for a hypothetical mission to the northern lowlands at approximately 35°N, 320°E (which is within a gray area in Figure 3.6). The committee notes that this is not intended to be comprehensive or universally appropriate; the NASA Planetary Protection Office (PPO) would need to perform more detailed analyses with relevant variables for each NASA mission being categorized. This estimate uses a 15-hour time period (an extended sol daytime period) as the duration under which terrestrial biota could survive UV exposure under Martian conditions. This duration is shorter than the conservative estimate (1-2 sols) adopted in this Chapter but is used here as a compromise with the knowledge that Deinococcus bacteria, renowned for UVC resistance, are not expected to survive longer than on order of an hour at mid-day on Mars. Drawing from observations of average wind speeds measured by Viking Lander 2 (~10 m/s was the upper range for the daily mean [Martínez et al. 2017]), Phoenix (~6-10 m/s [Holstein-Rathlou et al. 2010]), and Mars Science Laboratory (~<10 m/s during the Bagnold Dune wind characterization campaign, for ~18 sols during winter [Martínez et al. 2017; Newman et al. 2017]), and assuming that the hypothetical northern lowlands

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¹³ See Daubar et al. (2013) and the updated catalogue at Daubar et al. (2020).

¹⁴ Alfred McEwen, personal communication.

location has similar wind conditions to the VL2 and Phoenix landers, this estimate uses a wind speed of 10 m/s. At this velocity a suspended particle could travel approximately 540 km in 15 hours. Adding margin for higher wind speeds, the committee adopts a buffer radius of 600 km. This is considered a conservative value because it assumes unidirectional transport and does not include dust settling time. Landing during atmospheric conditions that promote higher wind speeds (e.g., dust storms) would necessitate a larger buffer zone; the committee’s calculation here assumes normal conditions. Because caves represent radiation-shielded environments where terrestrial biota could potentially proliferate, a conservative buffer distance is appropriate. As measurements and models of wind transport of dust and aerosols on Mars are improved, estimates of appropriate buffer zone sizes can also be improved.

Box 3.2 summarizes the committee’s principal conclusions in response to the charge from NASA.

RELAXED BIOBURDEN REQUIREMENTS

The previous section of this report noted that the current state of knowledge of Mars, and particularly its subsurface conditions, lacks important key information. Present thermal, radar, and imaging data are often limited by low spatial resolution and coverage. Identification of candidate subsurface access points is limited at the present to those >25 m across. Consequently, much of the current understanding of the subsurface depends on models rather than observations. The risk of harmful contamination cannot be dismissed, even for the regions outlined in Box 3.2, where lander bioburden requirements could be relaxed, but not removed. The committee interprets a designation of relaxed bioburden standards for some Mars landed missions to be consistent with a Category IV mission for which the maximum surface bioburden is less stringent than specified in current NASA and COSPAR policy but not equivalent to Category II, because there will still be some explicit approach to ensuring appropriate spacecraft cleanliness.

Provisions for mitigating remaining risks could be satisfied using standard aerospace industry cleanliness standards and practices even though they would be less stringent than the NASA and COSPAR requirements now required for Category IV. For example, following established aerospace industry cleanliness standards and practices (such as IEST-STD-CC1246E¹⁵) that are used to maximize product reliability could equally ensure cleanliness for relaxed planetary protection bioburden missions. Aerospace industry standards provide a uniform method for specifying product cleanliness levels and implementing a contamination control program as part of product/mission assurance.

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¹⁵ Military Standard 1246C, providing a basis and a uniform method for specifying product cleanliness levels and contamination control program requirements, as an example of standards and practices in use.