A Lunar Microbial Survival Model for Predicting the Forward Contamination of the Moon

2.1. Definition of “lethal dose”

We define the term lethal dose (LD) here to mean reducing a viable bioburden by a factor of −10 logs (10⁻¹⁰) in which the kill rate of a given factor is empirically derived for a reduction of −6 logs from time zero to some specific time step and dose, followed by theoretically extrapolating an additional −4 logs reduction in microbial inactivation. A LD per biocidal factor is considered the point at which the bioburden is “functionally sterilized” by that biocidal factor. Furthermore, most kill kinetics for biocidal factors follow exponential decay curves in which significant die-offs are observed over short time periods (first phase) followed by slower additional die-offs (second phase) due to shielded or aggregated spores or cells being protected from the biocidal factor. To simplify the overall LMS modeling, we will use two linear models to represent the two phases of the kill curves reported here and from the literature.

2.2. Estimating the accumulated microbial bioburdens on the Moon

Since the Luna 2 spacecraft impacted the Moon on September 14, 1959, 54 missions have either landed or crashed 77 vehicles or major components on the lunar surface (Table 1). No systematic estimate of the microbial bioburdens on all of these vehicles has been compiled. A study by Dillon et al. (1973) did survey the literature to estimate the bioburdens at launch for vehicles launched before February 1971. Based on the report by Dillon et al. (1973), spacecraft bioburdens are estimated for all documented vehicles beginning with Luna 2 in 1959 and extending to the Lunar Atmosphere Dust and Environment Explorer (LADEE) spacecraft that impacted the lunar surface on April 18, 2014 (Table 1). Data for spacecraft specifications including landing or impact dates, latitude and longitude coordinates of landing or impact sites, and vehicle dry masses were derived from the Lunar and Planetary Institute website (www.lpi.usra.edu/lunar/missions), Apollo by the Numbers (Orloff, 2000), Catalog of Apollo Experiment Operations (Sullivan, 1994), and The International Atlas of Lunar Exploration (Stooke, 2007).

Estimates of the probable bioburdens at landing (Table 1, column 10) for the LMS model of all spacecraft were calculated based on 16 sequential model assumptions (A).

(A1) A key assumption of the LMS model is that microbial cells and spores occur on spacecraft surfaces as single cells or small aggregates of cells/spores such that each cell or spore is directly exposed to the space conditions in question. Large aggregates of 10's to 100's of cells are assumed to be absent. This assumption is supported by the literature on microbial surveys of spacecraft surfaces in which colonies on assay plates generally appear as low densities of single colonies per assay plate (e.g., Schwendner et al., 2013).

(A2) The data of Dillon et al. (1973) on the number of culturable bacteria (henceforth culturable bioburden) at launch for lunar spacecraft from 1959 to February 5, 1970 were reported as originally published (Table 1, column 7). The review by Dillon et al. (1973) was consistent with other studies on microbial bioburdens at launch for lunar spacecraft (Powers, 1967; Puleo et al., 1970, 1973a, 1973b; Favero, 1971; Taylor et al., 1973; Herring et al., 1974).

(A3) Lunar Orbiters 1 and 2 were assigned the launched bioburden of 2.74 × 10⁵ viable cells based on the mean of bioburdens reported for Lunar Orbiters 3, 4, and 5 (Table 3 in Dillon et al., 1973).

(A4) All Apollo surface payloads landed on the Moon were assigned bioburdens of 7.01 × 10⁵ viable microbes at launch based on similarities to the Surveyor spacecraft in designs, complexities, masses, and the mean culturable bioburdens published for Surveyors I through VII (Table 3 in Dillon et al., 1973).

(A5) Luna 18, 19, 20, 21, 22, 23, and 24 were assigned bioburdens of 4.0 × 10⁷ at launch based on similarities to other Luna spacecraft in design, complexity, mass, and the mean bioburdens per launched lander (Table 5 in Dillon et al., 1973).

(A6) Bioburdens at launch for the Saturn S-IVB boosters that were crashed into the Moon after Apollo 13, 15, 16, and 17 missions were assigned the value of 3.1 × 10⁸ viable microbes at launch based on the published bioburden for the Apollo 14 S-IVB booster (Table 7 in Dillon et al., 1973).

(A7) Apollo 15, 16, and 17 Lunar Module (LM) descent stages were assigned culturable bioburdens of 3.9 × 10⁷ viable cells based on the published bioburdens for Apollo 11, 12, and 14 LM descent stages (Table 7 in Dillon et al., 1973).

(A8) Apollo 11, 15, 16, and 17 LM ascent stages were assigned the bioburden value of 1.4 × 10⁷ viable cells based on the average of LM ascent stage bioburdens for Apollo 12 and 14 (Table 7 in Dillon et al., 1973).

(A9) Lunokhod 1 and 2 were assigned the culturable bioburdens of 9.6 × 10⁶ viable cells based on the mean bioburdens at launch for all Luna missions (4.0 × 10⁷; Table 5 in Dillon et al., 1973) scaled downward by a factor of 0.24 to adjust to the lower masses of Lunokhod 1 and 2 (Table 1; this study). The average dry mass of all Luna missions was 3384 kg, and the average dry mass for the Lunokhod 1 and 2 rovers was 795 kg. The Lunokhod rovers had in general one-quarter the mass of the Luna landers and, therefore, we assigned one-quarter the culturable bioburdens (i.e., on a dry mass basis).

(A10) Apollo 15 and 16 subsatellites were assigned bioburden values of 1.59 × 10⁴ viable cells based on the average value for Lunar Orbiters 3, 4, and 7 (Dillon et al., 1973) adjusted for mass differences. The mean dry mass of Lunar Orbiters 3, 4, and 7 was 387 kg and the mean dry mass of two Apollo subsatellites was 41 kg (Table 1; this study). The Apollo 15 and 16 subsatellites had in general only 0.11 mass of the Lunar Orbiters, and thus, we assigned only 0.11 of the bioburdens (i.e., on a dry mass basis).

(A11) Launch estimates for spacecraft contamination between April 10, 1993 (Hiten orbiter) and April 18, 2014 (LADEE orbiter) were not readily available. The vehicles between these dates in Table 1 (column 8) were assigned bioburdens at launch based on taking the average bioburden per kg per spacecraft (2.54 × 10⁴ spores–cells/kg) (Dillon et al., 1973) and estimating each value in Table 1, column 8, by scaling the average bioburden/kg dry mass of published spacecraft to the dry masses of the more recent spacecraft.

(A12) Early literature on the culturable bioburdens on spacecraft (e.g., Powers, 1967; Puleo et al., 1970; Favero, 1971; Dillon et al., 1973; Herring et al., 1974) underestimated the total bioburdens at launch due to the emphasis on enumerating heterotrophic aerobic spore-forming and nonspore-forming bacteria. A paradigm of modern molecular microbiology states that between 1% and 0.01% of microbial species in natural ecosystems are culturable (Ward et al., 1992; Amann et al., 1995; Torsvik et al., 1996). However, recent studies on the microbial diversity of spacecraft surfaces have identified only a small number of species that are generally considered nonculturable (Venkateswaran et al., 2001; La Duc et al., 2004). Unlike natural soil and marine ecosystems that are rich in water and nutrient resources, spacecraft are oligotrophic ecosystems that are likely to be constrained in nonculturable species. Thus, a conservative scalar of 2 logs (10²) upward for data in Table 1, columns 7 and 8 was used to include under-represented and nonculturable microbial species present on spacecraft surfaces. The data given in Table 1, column 9, for all lunar spacecraft are reasonable estimates of the total probable bioburdens at launch for each mission.

(A13) Apollo LM ascent stages that were later impacted on the surface of the Moon were scaled an additional 2 logs upward based on the increased bioburdens contained within the astronaut habitats. In general, microbial contamination can be between 10² and 10³ higher within human crewed compartments than in robotic vehicles (Puleo et al., 1973b).

(A14) Lunar cycles (henceforth lunations) in Table 1 were estimated from dates that spacecraft were landed or crashed on the surface to January 1, 2019 based on a lunation equal to 42,524 min (29.53 days) for noon-to-noon cycles.

(A15) No articles in the literature were found that quantitatively estimated the loss of spacecraft bioburdens due to impact processes on the lunar surface. However, a number of studies (e.g., Fraser et al., 1971; Whitfield et al., 1973; Burchell et al., 2004; Barney et al., 2016; Plescia et al., 2016) have demonstrated that compression forces on bacteria during simulated impact events into regolith-like materials yield a range of biocidal effects with an overall average of approximately −4 logs (10⁻⁴) in survivors. Thus, all spacecraft crash landed on the Moon were scaled downward by −4 logs to accommodate the probable loss of viable bacteria during impact events (adjusted in Table 1; column 10 data).

(A16) Although recent articles (e.g., Glavin et al., 2010; Loftus et al., 2010; Linnarsson et al., 2012) have examined the biotoxicity of lunar dust, we have assumed that spacecraft microorganisms were not mixed with lunar regolith, and thus, the potential biocidal effects of lunar dust are ignored in the current LMS model.

2.3. Estimating the inactivation rates of the landed or crashed bioburdens on the Moon

2.3.1. Inactivation of microorganisms exposed to high vacuum

A number of articles have studied the effects of various extraterrestrial factors on microbial survival (e.g., Bücker et al., 1994; Horneck et al., 1994, 2010; Schuerger et al., 2003), but most studies report only a few time steps for test microorganisms under individual stress factors. To develop a first-order LMS model on how fast terrestrial microorganisms might be inactivated on the Moon by cis-lunar vacuum conditions, control data from a diversity of articles were plotted against time for B. subtilis, a spore-forming bacterial species (Fig. 1). Data from “control” treatments refer to survival rates for B. subtilis spores maintained at ∼25°C and exposed to pressures between 1 × 10⁻⁴ and 6 × 10⁻⁷ Pa. The data were extracted from articles published from microgravity and high-vacuum laboratory experiments. Effects of other parameters on microbial survival in the literature were temporarily ignored. Data for vacuum effects on B. subtilis were derived from the studies of Bücker et al. (1974), Dose and Klein (1996), Hagen et al. (1971), Horneck et al. (1984, 1994, 1995), Koike and Oshima (1993), Lorenz et al. (1968), and Morelli et al. (1962).

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FIG. 1.

Effects of vacuum only on the survival of Bacillus subtilis spores. Data were extracted from the cited articles and represent the control treatments in the studies that were exposed to only high vacuum conditions during microgravity or laboratory experiments. Linear models were independently generated for both phases with each point representing the mean of the appropriate experimental replicates summarized in the articles. The longest time step is 2107 days for B. subtilis spores exposed to space on the LDEF (Horneck et al., 1994). The precise inflection point (or elbow) between the first and second phases occurred at −0.473 logs (y-axis) and 93.7 days (x-axis). LDEF, Long Duration Exposure Facility.

2.4. Statistics and data management

Data for Fig. 1 were derived from the articles indicated in the figure and these represent the mean values for the vacuum-only controls in each study. Data for Fig. 2 were based on the experiments for biocidal effects of elevated temperatures already described. Data for Fig. 3 were based on the experiments for interactive effects of high temperature (100°C) and low pressure (10⁻⁶ mbar) already described. Data for Fig. 4 were adapted from a previous study by Schuerger et al. (2006) on the effects of UV irradiation on B. subtilis HA101 spores. However, we have reanalyzed the data from the study of Schuerger et al. (2006) to force a spline point where the two linear models overlap. We present here updated linear models for the data shown in Fig. 4.

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FIG. 2.

Kill curves for the inactivation of B. subtilis HA101 spores exposed only to high temperatures for up to 56 days at laboratory normal pressures of 101.3 kPa. Linear models were independently generated with each datum point representing four replicates in separate and completely randomized experiments. Each time step was conducted twice (total n = 8 per day tested) and the means of both experiments per time step were plotted (i.e., each plotted point represents n = 4 samples). Asterisks represent time steps in which no viable spores were detected in the assays (lower detection limit = 11 viable spores per sample). The time steps marked by na were not assayed.

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FIG. 3.

Interactive effects of high temperature (100°C) and low pressure (10⁻⁴ Pa) on spore survival by B. subtilis HA101. Spores exposed to 10⁻⁴ Pa and 25°C decreased approximately −0.25 logs compared with laboratory controls at 101.3 kPa and 25°C, whereas spores exposed to 101.3 kPa and 100°C decreased approximately −2 logs. *The combination of high temperature and low pressure resulted in the sterilization of the aluminum coupons doped with bacterial spores. The minimum detection level was 11 spores per sample. Data were log transformed and subjected to ANOVA and protected least-squares mean separation tests; different letters indicate a significant difference between means (p ≤ 0.05; n = 12). Error bars are standard deviations of the means. ANOVA, analysis of variance.

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FIG. 4.

Kill curve kinetics for B. subtilis HA101 spores exposed to UV irradiation and exposed at normal laboratory pressures of 101.3 kPa. The data are replotted from Schuerger et al. (2006) with updated linear models. In the original article, the plotted data represented the means of eight replicates. Linear models were independently generated. The first phase of the kill curve represents a rapid inactivation of −4 logs over the course of only 2 min. The second phase of the kill curve exhibits a much slower inactivation rate that decreased an additional −2 logs between 2 and 30 min of UV exposure. The precise inflection point (or elbow) between the first and second phases occurred at −4.196 logs (y-axis) and 1.09 min (x-axis). UV, ultraviolet.

Linear models for Figs. 1, 2, and 4 were independently generated with either SigmaPlot (v13.0) or Microsoft Excel for Mac 2011 (v.14.7.3) graphing subroutines. Statistics were conducted with the PC-based Statistical Analysis System (SAS) version 9.2 (SAS Institute, Inc., Cary, NC). Data for Figs. 1, 2, and 4 were log10-transformed and analyzed by linear regression using the SAS subroutine PROC REG. All linear models for Figs. 1, 2, and 4 exhibited significant p-values (p ≤ 0.05). Data for Fig. 3 were log10-transformed and subjected to an analysis of variance and a protected least-squares mean separation test (PROC GLM; p ≤ 0.05).

Untransformed and original data sets for Figs. 1–4 are presented in Supplementary Tables S1–S4, respectively, with annotations (Supplementary Data are available online at https://www.liebertpub.com/suppl/doi/10.1089/ast.2018.1952). Furthermore, we include the LMS model code (Supplementary Table S5), used to derive Figs. 5 and 6.

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FIG. 5.

Bioburden reductions for the strongest biocidal factors on external surfaces. The top panel shows the reduction due to temperature and vacuum acting together, the middle panel shows UVC and UVB irradiation acting on a flat level surface. The bottom panel combines all factors. The color indicates the instantaneous reduction rate expressed in logs per lunation, in which larger values indicate the greatest bioburden reductions. The smaller panels at right integrate the values in the left panels over the entire lunation. The primary phase of UV irradiation on survival is represented by the faint-blue vertical bar at sunrise (i.e., 0.25 fractional lunations). The combination of temperature, vacuum, and UV generates at least −10 log reductions in spacecraft bioburdens in less than a single lunation for latitudes equator ward of 89.8°.

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FIG. 6.

The LMS model applied to all lunar landings from the moment of touchdown to January 01, 2019. The upper left panel shows the bioburden at landing per spacecraft. Subsidiary panels show the loading on external surfaces (upper right), internal surfaces in thermal contact with the exterior (lower left), and deep insulated interior surfaces (lower right) over time. As of January 01, 2019, the exterior surfaces of all landed spacecraft are currently sterile, whereas some spores remain in the deep interiors of more recently landed spacecraft, particularly at polar latitudes. LMS, Lunar Microbial Survival.

3.1. Estimated bioburdens on launched spacecraft to the Moon

Since September 14, 1959, 54 lunar missions have delivered 77 spacecraft, boosters, payloads, rovers, and other structures to the surface of the Moon. Using a 16-factor model, we estimate the probable viable bioburdens on all spacecraft components that have reached the lunar surface. Overall, 4.57 × 10¹⁰ viable microorganisms are likely to have been landed or crashed on the lunar surface (Table 1). The average microbial bioburden per individual spacecraft was estimated to be 5.47 × 10⁸ viable vegetative cells and spores per spacecraft. Dividing the average spacecraft bioburden (5.47 × 10⁸ cells/spores) by the average dry mass (2375 kg) of the spacecraft and structures landed or crashed on the Moon yields an estimate of 2.30 × 10⁵ viable cells/spores per kg. The highest estimated bioburden was predicted for the Apollo 14 LM descent stage at 1.0 × 10¹⁰ spores/cells at lunar contact. A number of lunar landers and other Apollo LM descent stages were also predicted to have >1.0 × 10⁹ spores/cells at lunar contact.

Although the human occupied LM ascent stages initially had significantly higher bioburdens within the crewed habitats compared to other spacecraft components (Table 2, column 9), all LM ascent stages were crashed into the lunar surface after serving to return the astronauts to the Apollo Command Modules in lunar orbit. Based on the LMS model assumption A15 that a bioburden reduction of −4 logs was achieved by impact compressional forces, the average total probable bioburden per crashed LM ascent stage was estimated to be 7.57 × 10⁶ surviving cells/spores at lunar impact.

3.2. Pressure effects on microbial survival

A biocidal kill curve for B. subtilis spores exposed to vacuum (<10⁻⁴ Pa) from <1 day (e.g., 90 min) (Bücker et al., 1974) to almost 6 years (e.g., 2107 days) on the LDEF (Horneck et al., 1994) in LEO yields a biphasic kill curve in which a rapid decrease (∼70%) can be observed for the first 94 days followed by a much slower kill rate out to 2107 days (99%; −2 logs) (Fig. 1). Although temporal kill kinetics are in general lacking for nonspore-forming species under simulated or actual spaceflight conditions, a few studies have demonstrated that nonspore-forming species are significantly more sensitive to the biocidal effects of space conditions (e.g., Hagen et al., 1971; Koike, 1993; Koike and Oshima, 1993; Horneck et al., 2010). Based on these few studies, we assume here that nonspore-forming bacteria will be inactivated at a rate of ∼10 × faster than the kill kinetics for B. subtilis spores.

3.3. Long-term exposure of B. subtilis spores to high temperatures

Thermal infrared photons in solar irradiation can raise the temperatures of external spacecraft surfaces to >100°C during each lunation (Table 3). For example, thermocouples on Surveyors I, III, and V recorded temperatures >100°C for 4–5 days per lunation (Lucas et al., 1966, 1967a, 1967b), with the highest spacecraft surface temperature recorded at 140°C for the Surveyor V planar array antenna (Lucas et al., 1967b). Furthermore, through conduction and radiant heat transfer, many internal components of landed equipment or spacecraft will reach between 70°C and 90°C during each lunar cycle. Table 1 demonstrates that spacecraft on the lunar surface have experienced between 57 and 737 lunations since landing or crashing on the surface (based on dates estimated from landing to January 1, 2019). Although no estimates on thermal inertia or thermal conductivity within spacecraft structures on the Moon were found in the literature, we assume here that ∼50% of all internal components of lunar spacecraft are likely to achieve temperatures that exceed 70°C during the lunar day, and that perhaps as much as 25% of all spacecraft internal components may exceed 100°C.

The thermal inactivation models of Dillon et al. (1973) predict thermal inactivation of all external surfaces of lunar spacecraft between 9 and 12 months after landing. To validate the thermal inactivation models of Dillon et al. (1973), and extend the models to lower temperatures, a series of laboratory experiments were conducted to test the survival of the high-temperature-resistant spores of B. subtilis (Hagen et al., 1971; Horneck et al., 1984) at elevated temperatures between 70°C and 100°C. Results indicate that at 100°C, as little as 2 Earth days were required to reduce the viability of B. subtilis spores by −6 logs (i.e., below the detection limit of the assay) (Fig. 2). At 90°C, between 4 and 7 days were required to induce a decline of −6 logs in the number of viable spores recovered from coupons. At lower temperatures of 70°C or 80°C, the populations of B. subtilis spores decreased between −4 and −3 logs, for 14 and 56 days, respectively. Although the 70°C or 80°C assays were not continued to extinction due to logistical constraints of the experiments (e.g., time and resources), we assume the cells would have continued to lose viability with longer incubation times at the same rates as shown in Fig. 2. The extrapolation of thermal decay curves for microorganisms to time steps beyond the available empirical data, as used here, is consistent with other literature on thermal inactivation rates of Bacillus spp. under diverse humidity and high-temperature regimes (e.g., Nicholson, 2003; Schubert and Beaudet, 2011).

3.4. Interactive effects of low pressure and high temperature

Although temperatures >90°C are likely to inactivate greater than −6 logs of Bacillus spores per lunation (Fig. 2), most spacecraft and deployed equipment are vented to the vacuum of space, and thus, cis-lunar vacuum will likely interact with temperatures >70°C to induce significant bioburden reductions within internal surfaces. A series of short-term assays were conducted to determine whether the interactive effects of low pressure (<3 × 10⁻⁴ Pa) and high temperature (100°C) would increase the lethality of the simulated lunar surface. When spores were incubated for 8 h at 25°C and 101.3 kPa in the laboratory (control), ∼3.0 × 10⁶ spores per coupons were recovered (Fig. 3). When spores were incubated for 8 h under low pressure only or high temperature only conditions, survival was reduced by −0.5 and −1 logs, respectively. In contrast, when spores of B. subtilis were incubated for 8 h under the combined conditions of low pressure (<10⁻⁴ Pa) and 100°C, no viable cells were recovered from the aluminum coupons (i.e., greater than −6 logs of spores were killed by the combined treatments). Thus, the synergistic effects of low pressure and high temperature resulted in at least −6 logs of increased lethality on the deposited spores. During the course of each lunation, the shallow internal and external temperatures of Sun-exposed compartments will likely be >100°C for 1–6.5 days (Table 3).

3.5. Solar UV irradiation effects on microbial survival

Figure 4 reanalyzed a subset of the data from a previous study on the effects of UV irradiation on the survival of seven Bacillus spp. For simplicity sake, and to be aligned with the data shown in Figs. 1–3, we use only the kill curve for B. subtilis HA101 (adapted from Schuerger et al., 2006). The kill curve is split into a biphasic model in which the first phase shows a rapid decrease of −4 logs of inactivated spores over a period of 2 min, followed by a much slower degradation rate of the second phase that required 30 min to achieve a bioburden reduction greater than −6 logs per coupon (Fig. 4). The transition (i.e., spline point) between the two linear phases shown in Fig. 4 is located at log₁₀ (y) = −4.196 and (x) = 1.09 min. The UVC+UVB flux that killed >10¹⁰ spores of B. subtilis was estimated to be 57.9 kJ/m² by Schuerger et al. (2006), which is delivered to the equatorial lunar surface in 36 min at local noon (Table 2).

3.6. Ionizing radiation effects on microbial survival

In scenarios in which microorganisms are not shielded, SWPs will reduce efficiently the amount of spores with an LD rate of ∼100 days to achieve survival of −10 logs (Table 2). In contrast, due to the very low energies of the SWPs, there will be no effect below a depth of ≥300 μm of shielding material, and the contribution of SPEs becomes dominant. Calculations for the annual mean dose behind a shielding depth of ∼3.7 mm aluminum (which equals the mean shielding depth of an astronaut EVA suit) show dose values ∼3 Gy per year from SPEs and, thus, >1600 years are needed to approach a survival of −10 logs. SPEs, therefore, cause no significant reduction of microorganisms. The annual absorbed dose delivered by GCRs is ∼0.1 Gy, and thus >1.5 × 10⁴ years are needed to reach the required level of −10 logs. GCR particles, therefore, contribute very little to the inactivation of spores in terms of absorbed dose over realistic time scales.

Taking into consideration inactivation studies of B. subtilis spores hit by a single HZE particle, bacterial spores are faced with a much higher inactivation probability. Applying the measured inactivation cross sections of 5.24 μm² observed in the Biostack spaceflight, HZE particles might cause an inactivation of −10 logs after ∼180 years (Reitz et al., 1995, Horneck et al., 1989; Reitz, 2008) (Table 2). In contrast, when using inactivation cross sections measured in accelerator experiments of only 1.41 μm², a survival rate of −10 logs is achieved after >2600 years.

The only significant LD induced by ionizing radiation was observed for routine SWPs impinging on external spacecraft surfaces. In Table 2, we modeled that only −3 log reductions would be possible for SWPs on external surfaces of spacecraft per lunation. The other types of ionizing radiation are expected to have minimal effects (e.g., −0.0045 logs per lunation for HZE particles), or no adverse effects per lunation for SPEs, SCRs, GCRs, or secondary particle fluence rates.

3.7. LMS model

The LMS model combines together all the deleterious effects described in Figs. 1–4 and Table 2 to predict the number of viable microbial spores and vegetative cells on spacecraft surfaces at any time after landing on the Moon's surface. In this way, the LMS model is useful not only to describe the bioburden present on past spacecraft but may also be used to assess the time required for bioburdens to be deactivated on future spacecraft, not yet flown. The text that follows will detail how each deleterious effect is considered mathematically, and how together each of these effects builds up the overall LMS model.

To capture the protective properties of spacecraft hardware, the LMS model divides the bioburden residing on spacecraft into three categories: (1) spores residing on the exterior surfaces of spacecraft that are subject to the greatest variations in temperature, solar UV, SWPs, and all other effects without shielding; (2) spores residing on the shallow interior of spacecraft but that are in thermal contact with the spacecraft exterior surfaces; and (3) spores residing deep within the spacecraft and thermally insulated from the spacecraft exterior surfaces heated directly by solar irradiation. Thus, spores in the second category are considered shielded from all deleterious effects except for vacuum and heat, whereas those in the third category are subject only to the long-term effects of cis-lunar vacuum. To provide upper bounds on survivability, the code makes the assumption when considering each category that the entire bioburden described in Table 1 (5.47 × 10⁸ spores/cells per spacecraft) can be found within that category.

4.1. LMS predictions for current landed or crashed spacecraft on the Moon

A LMS model was developed that can be used to predict spacecraft bioburdens at landing, and to estimate inactivation rates for combinations of four biocidal factors present on the lunar surface. The primary reasons for developing the LMS model were to predict whether previously landed spacecraft might harbor viable bioburdens over time and identify for Earth return the most promising landed or crashed spacecraft hardware for future human missions. In addition, the LMS model can be applied to a diversity of lunar astrobiology studies (see Gronstal et al., 2007; Crawford et al., 2012) including research on microbial survival of landed or crashed spacecraft, field testing planetary protection strategies for mitigating the presence of microbial bioloads on interplanetary spacecraft, and technology development to advance the human exploration of the Moon and Mars.

The LMS model was based on predicting the spacecraft total probable bioburdens at lunar contact, and then estimating the inactivation rates of bacteria by UV irradiation, vacuum, high temperature, and ionizing radiation on the lunar surface. Spacecraft bioburdens at launch (Table 1) were estimated by using published data for a significant number of lunar missions up to 1971 (column 7; Dillon et al., 1973), followed by reasonable extrapolations of later missions based on assumptions A1 to A11. The data were scaled upward by two orders of magnitude to include (1) nonculturable bacterial species, (2) the likely presence of low numbers of fungi, archaea, actinomycete, and other extremophile species on spacecraft (see the literature in assumption A12, and Stieglmeier et al., 2009; Ghosh et al., 2010), and (3) microbial communities associated with humans in Apollo LM ascent stages (assumption A13). The LMS model predicted the total viable bioburden at lunar contact of 4.57 × 10¹⁰ propagules for all spacecraft, and the average per vehicle bioburden of 5.47 × 10⁸ spores/cells (Table 1).

The LMS biocidal inactivation rates for vacuum, UV irradiation, high temperature, and ionizing radiation were focused solely on the kill kinetics for Bacillus spp. because of inadequate data on nonspore-forming bacteria and fungi in space. However, because Bacillus spores are significantly more resistant than nonspore-forming species to many of the conditions found in interplanetary space (see Horneck et al., 2010), we assume that the inactivation of Bacillus spores will be significantly slower than for all other microorganisms present on spacecraft. Thus, if B. subtilis spores are killed at the rate of −10 logs per biocidal factor (the LD dose used here) for any given time step, then all other nonspore-forming species are assumed to be killed. For example, the nonspore-forming bacteria, Micrococcus sp. and Staphylococcus epidermidis, exhibited significantly lower survival rates of −1 and −2 logs, respectively, compared with only −0.25 logs for B. subtilis var. niger spores when exposed to ultrahigh vacuum close to 10⁻⁸ Pa at 60°C (Hagen et al., 1971).

The slowest inactivation rate for spacecraft microorganisms was found to be due to the effects of high vacuum (down 10⁻⁸ Pa; see literature cited in Fig. 1) yielding −0.02 logs per lunation, or −0.247 logs per year and −2.47 logs per decade. The LMS model also assumes that all surfaces on and within the landed spacecraft are exposed to the cis-lunar vacuum, with the exception of sealed tanks and tubing for cryogenic fluids. Thus, even if microorganisms are deeply embedded within landed or crashed vehicles, survival of microorganisms will be negatively affected by low pressure alone. Interestingly, the data from the cited articles in Fig. 1 were closely aligned with the linear models (i.e., low scatter), and the data were derived from multiple strains of B. subtilis in experiments that extended over a wide range of low pressures (10⁻⁴ to 10⁻⁸ Pa) in assays that were conducted in terrestrial laboratories, LEO, and cis-lunar space.

Both UV irradiation and high temperature alone were predicted to contribute aggressive kill rates for microorganisms on external surfaces with up to −2290 logs or −97 logs of inactivation, respectively, at equatorial latitudes. As the latitudes increased, the corresponding effects would be reduced (Table 2). The most aggressive kill rates were predicted for UV irradiation on external surfaces. Next, synergistic interactions of high temperature and high vacuum in which both external and shallow internal surfaces of spacecraft would be expected to see up to −188, −116, or −16 log reductions for latitudes at 0°, ± 30°, or ±60°, respectively. Only a few articles were found in the literature that tested the interactive effects of temperatures <100°C and pressures close to cis-lunar vacuum (≤10⁻⁸ Pa). For example, using both spore-forming (B. subtilis var. niger) and nonspore-forming (Micrococcus sp. and S. epidermidis) bacteria, Hagen et al. (1971) demonstrated that simultaneously increasing temperatures from 25°C to 60°C in a high vacuum environment of 10⁻⁸ Pa resulted in increased inactivation of the simulated space environment for all three bacteria. The smallest reduction in surviving spores was observed for B. subtilis (−0.25 logs), but also nonspore-forming species exhibited −1 to −2 log reductions over the same temperature range.

Although only the synergistic effects of temperatures at 100°C and low pressures at 10⁻⁴ Pa were tested here, other literature (Horneck, et al., 1984, 1995; Dose and Klein, 1996; Nicholson et al., 2000) indicates that the interactions of UV+vacuum can increase the lethality of the space environment by upward of two additional orders of magnitude. In short, the current LMS model is likely underestimating the lethality of the space environment for microbial spores/cells on spacecraft surfaces due to the periodic occurrence of temperatures >100°C on spacecraft hardware (e.g., Surveyor III compartments periodically reached 140°C during the lunar day) (Table 3; Lucas et al., 1967a), by interactive effects of vacuum and lower temperatures than tested here, and by the interactive effects of UV+vacuum.

When considering the effects of various types of ionizing radiation on microbial survival on the Moon, only SWPs were found to contribute any significant biocidal effect (Table 2). However, even for SWPs, the inactivation rate was only −3 logs per lunation, and for only the external surfaces of the spacecraft. Microbial survival rates on all internal surfaces, whether shallow or deeply embedded within the spacecraft, were found to be relatively unaffected by all forms of ionizing radiation so as to be regarded here as negligible in the LMS model. This finding is based on the high dose rates reported in the literature for B. subtilis that approached 3 kGy for a −6.0 log reduction (e.g., Moeller et al., 2007, 2010), and that were extrapolated here to 5 kGy for −10 logs (based on 450 Gy per −1.0 logs inactivation) (Moeller et al., 2007, 2010).

The LMS model overall predicts that most spacecraft listed in Table 1 would be expected to have zero viable bacteria as of January 1, 2019 (Fig. 6), except for spacecraft landed in polar regions and within the past 13 years. Table 4 presents the results of two LMS model runs for the last 10 landed or crashed spacecraft on the Moon for January 1, 2019 and January 1, 2030. All 10 of these spacecraft are likely to still harbor low numbers of viable bacteria as of January 1, 2019. However, LMS predictions for 2030 indicate that only the Chang'e/Yutu lander is likely to retain any significant numbers of viable bacteria, and only on deeply embedded surfaces.

The only other microbial survival model found in the literature for microorganisms on lunar spacecraft was advanced by Dillon et al. in 1973 (henceforth the Dillon model). The LMS model predicts overall significantly longer survival times than the Dillon model for microorganism on deeply embedded spacecraft surfaces on the Moon because the Dillon model only considered the effects of thermal inactivation kinetics extrapolated to all spacecraft surfaces, which we have shown to only be persistent on external and shallow internal surfaces. The Dillon model predicted that all microorganisms would be killed within 7–8 months. In contrast, the LMS model separates thermal effects into external surfaces, shallow internal surfaces, and deeply embedded internal surfaces, and includes four biocidal factors in the model (i.e., UV irradiation, high temperature, vacuum, heat+vacuum, and ionizing radiation) instead of only just thermal effects. If we compare the inactivation of microorganisms between the Dillon and LMS models for external surfaces only, the LMS model predicts up to a −97 log reduction for one lunation (14.77 days for daylight hours) on spacecraft near the equator (Table 2), which is substantially faster than the 7–8 months in the Dillon model. The LMS model updates and refines the earlier Dillon et al. (1973) predictions by using four biocidal factors on the Moon instead of just thermal inactivation, and by considering external and internal surfaces separately. Thus, the LMS model predicts much faster inactivation of microorganisms on external surfaces and much slower death rates for internal surfaces than the Dillon model.

And finally, based on the definition that a −10 log reduction in viable bioburden equals a LD on the Moon, the LMS model predicts extremely high numbers of accumulated LD rates over the course of a given surface mission. For example, the overall LMS model for all spacecraft surfaces near the equator is −2479 logs per lunation. Such an inactivation rate equals an accumulation of 248 LDs per lunation, or as many as 1.82 × 10⁵ accumulated LDs for external surfaces on the Luna 2 impactor since September 14, 1959, the first spacecraft to make lunar contact (Table 1). Such high levels of microbial lethality on spacecraft surfaces all but preclude that most microorganisms on spacecraft will survive only very short periods of time on the Moon. Only deeply embedded internal surfaces offer adequate protection from thermal cycling, UV irradiation, and ionizing radiation on the Moon.

4.2. Validation of LMS model: the case of S. mitis and Surveyor III

During the Apollo 12 mission, astronauts Pete Conrad and Alan Bean landed the LM Intrepid within 500 m of the Surveyor III spacecraft that was present on the Moon for 942 days (31.9 lunations) from April 20, 1967, to November 20, 1969 (Nickle, 1971). During the surface EVA, the astronauts visited the Surveyor III spacecraft (Fig. 7A) and recovered ∼10 kg of components for return to Earth. The equipment was inserted into a nonsterile “beta-cloth bag” composed of “… a woven quartz-glass fabric covered with FEP Teflon …” on the lunar surface and kept in the beta-cloth bag until the samples arrived at the crew reception area (CRA) at the Lunar Receiving Laboratory (LRL) in Houston, TX (Nickle, 1971). It was at the CRA that the Surveyor III parts were then catalogued, photographed, and heat sealed in polyethylene bags until processed. Of the equipment returned to Earth, the TV camera (manufactured by Hughes Aircraft Company; HAC) and the electrical cables were assayed at the Manned Space Center, Houston, TX (renamed the Johnson Space Center in 1973) for microorganisms that may have survived 2.5 years on the lunar surface. The camera weighed 7.95 kg (Hughes Aircraft Company, 1970), and the cables are estimated here to be <0.25 kg based on the descriptions of the returned equipment by Nickle (1971).

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FIG. 7.

The Surveyor III camera (cam) was recovered by the Apollo 12 astronauts during a surface EVA in November 1969 and returned to Earth for analysis. (A) Surveyor III is shown in the foreground and the LM Intrepid is shown in the background. Surface thermocouples were placed on exterior walls of both Compartments A (ca) and B (not visible here; facing the LM). The astronauts handled the camera with unsterilized gloves during the EVA (see Section 4.2 for details). (B) The Surveyor III camera was photographed outside of a laminar flow hood during postflight processing of hardware. (C) Astronaut Pete Conrad and an unidentified photographer are seen adjacent to the TV camera in an open-door laboratory (background) that likely lacked filtered air. Images were downloaded from the website (www.apolloarchive.com/apollo_gallery.html). Images are courtesy of NASA: (A) NASA-JSC_A12-48-7100, (B) NASA-JSC_AP12-Surv-cam-noID, and (C) NASA-JSC_S-69-62290. EVA, extravehicular activity; LM, Lunar Module.

Mitchell and Ellis (1971) reported that a single pure culture of Streptomyces mitis was recovered from circuit board foam insulation that was deeply embedded within the camera body (Fig. 7B). The study used multiple replicates per each of three liquid media for the assays of samples from 33 locations on the external shroud (2 samples) and internal components (29 samples) of the camera. The materials tested (e.g., circuit boards, nylon ties, electrical wiring, and insulation foam) were either swiped with cotton swabs and the swab tips submerged in liquid culture medium, or materials were removed and placed directly into the enrichment medium. Of the 33 areas tested, only a single pure culture of S. mitis in one replicate for sample location 32 (foam insulation) was recovered. A similar set of 86 enrichment assays in which sections of electrical cables (e.g., nylon ties, insulation, wire, and teflon sleeving) were cut into short sections and submerged into multiple replicates per each of two separate liquid media yielded zero recovered microorganisms (Knittel et al. (1971). Although there were no prelaunch microbial assays conducted of the Surveyor III camera and cabling, an identical backup TV camera, designated the Type Approval Test (TAT) camera by HAC (Mitchell and Ellis, 1971), was assayed. The TAT camera was stored “… undisturbed in … bonded storage …” (Mitchell and Ellis, 1971) from before the launch of Surveyor III in April 1967 until the flight camera was processed for microorganisms in late 1969. The TAT camera served as a proxy for an Earth-based control and yielded strains of a Bacillus sp., several Aereobasidum spp., and Aspergillus pulvanis from internal electrical cabling.

The recovery of S. mitis from Surveyor III was reported by Mitchell and Ellis (1971) as the first known isolation of an Earth bacterium that had survived 2.5 years on the Moon. However, Rummel and colleagues (Glavin et al., 2004; Rummel, 2004; Rummel et al., 2011) have suggested that the isolate of S. mitis was likely a laboratory contaminant introduced into the assays during nonaseptic handling of the camera. For example, the camera was kept outside of laminar flow hoods for initial processing (Fig. 7B, C), including placing the camera on a desk in a generic laboratory with an open door clearly seen in the background (Fig. 7C). The laboratory appears to lack an active air filtration system, and the personnel appear in non-cleanroom garments.

Can the LMS model be used to support or reject the claim by Mitchell and Ellis (1971) that S. mitis survived 2.5 years on the Moon? First, if the launched culturable bioburden of Surveyor III was 1.97 × 10⁶ spores/cells (Dillon et al., 1973), the Surveyor III spacecraft weighed 296 kg at landing (dry mass) (Table 1), and the camera weighed 7.95 kg (Hughes Aircraft Company, 1970), then it follows that the landed culturable microbial bioburden for the TV camera alone would be ∼5.29 × 10⁴ spores/cells [i.e., ((1.97 × 10⁶)/296 kg) × 7.95 kg]. Second, although an hourly record of the internal temperatures of the TV camera is not available, the internal maximum temperature for the Surveyor III camera was estimated to be 70°C for lunar daylight hours per lunation (Mitchell and Ellis, 1971). Based on the temperature profiles of other spacecraft compartments on Surveyor III (e.g., Lucas et al., 1967a), maxima temperatures for any measured compartment were stable at the maximum temperatures for about one-third of the daylight hours per lunation.

Assuming that the internal components of the TV camera were close to 70°C for 33% of each lunar day per lunation, then the data shown in Fig. 2, (70°C), and Eqs. 3a and 3b, would predict that a population of spores of 5.29 × 10⁴ would be inactivated (<1 viable spore) after 10.4 lunations based exclusively on the biocidal effects of high temperatures at 70°C over time. Furthermore, if the full LMS model is applied to the same question, we estimate that a −188 log reduction would be expected for the shallow internal surfaces of the camera per lunation. Both answers are very much equal to a predicted level of zero surviving spores in the Surveyor III camera for the 2.5 years between landing and recovery.

Although we have not tested the interactive biocidal effects of vacuum (<10⁻⁴ Pa) and 70°C, we believe the enhanced lethality of low pressure and high temperature shown for 10⁻⁴ Pa and 100°C (Fig. 3) likely would hold for increasing the lethality of cis-lunar vacuum at 70°C. In addition, the electrical cabling assayed for culturable microorganisms (Knittel et al., 1971) would have been expected to contain ∼1.66 × 10³ culturable microorganisms at launch, and of these materials, the postmission assays yielded no recovered viable microbial spores or cells after 2.5 years.

The LMS model does not support the claim that the nonspore-forming bacterium, S. mitis, survived on the Moon for 2.5 years. The only plausible manner for S. mitis to survive for the 942 days on the lunar surface would be for the insulation foam to have been at significantly lower temperatures than 70°C encountered during each lunation. The LMS model also predicts that culturable bacteria should not be recoverable from the electrical cabling, as was described by Knittel et al. (1971). Based on the findings that viable bacteria and fungi were recovered from the ground control TAT camera (Mitchell and Ellis, 1971), only one out of several hundred enrichment assays was positive for any Earth microorganisms (Knittel et al., 1971; Mitchell and Ellis, 1971), we conclude that the lethality of the lunar surface is confirmed, and that the LMS model predicts the extremely low recovery rates of the microorganisms from the Surveyor III camera. We agree with Rummel and coworkers (Glavin et al., 2004; Rummel, 2004; Rummel et al., 2011) that the most likely explanation of the presence of S. mitis in the single positive culture tube from the assayed Surveyor III equipment was through contamination during the postlanding processing of the camera.

4.3. Introducing the terms lethal dose rate and functional sterilization

The LD rates for the four biocidal factors in the LMS model were based on new data presented here (i.e., high temperature alone [Fig. 2] and temperature+vacuum [Fig. 3]), or from the literature (i.e., vacuum alone [Fig. 1], UV irradiation [Fig. 4], and ionizing radiation [Table 2]). To make the LMS model easier to quantify, linear models were used when the data followed monophasic (Fig. 2) or biphasic (Figs. 1 and 4) responses. In addition, the effects of ionizing radiation on microbial survival were based on the linear decay rates for B. subtilis spores exposed to X-rays (e.g., Fig. 2 and Moeller et al., 2010).

Empirical bioburden reduction rates down to −6 logs can be easily derived in most laboratory experiments because starting populations are often fixed between 1.0 × 10⁶ and 5 × 10⁶ spores per sample (e.g., Schuerger et al., 2006; Moeller et al., 2010 and Figs. 2 and 4). As the initial populations are raised >5.0 × 10⁶ spores per sample (especially for spores dried on to spacecraft materials) (e.g., Schuerger et al., 2006; Moeller et al., 2010 and Figs. 2 and 4), the spores begin to develop multilayered aggregates in which buried spores are protected by spores in the upper layers of the aggregates (see Schuerger et al., 2005). Thus, the results are often skewed to higher survival rates at spore/cell populations >5.0 × 10⁶ per sample. The approach here for the LMS model was to use empirically derived data down to −6 logs, and then extrapolate an additional −4 logs for a total LD dosage of −10 logs of microbial inactivation.

We propose the term functional sterilization of a spacecraft surface to refer to the effects of biocidal factors acting to inactivate microbial spores or vegetative cells down to −10 logs. This approach is consistent with the Viking landers' heat-sterilization protocol (Puleo et al., 1977), in which the length of time to inactivate all spores and vegetative cells down to a probability of <10⁻⁴ below inactivating a known population of 10⁶ spores yields a total LD of −10 logs. The use of functional sterilization gives the astrobiology and planetary protection communities a pragmatic approach to determining when extremely low bioburden survival rates approach a functional level for spacecraft sterility, below which further bioburden reduction efforts appear unproductive in mitigating forward contamination any further.

4.4. LMS model caveats

Thermal transport models are required for complex spacecraft architectures on the Moon. First, the current LMS model idealized the spacecraft geometries as horizontal surfaces sitting parallel to the lunar terrain. However, more complex architectures will increase or decrease shading effects on other surfaces, making new LMS predictions more complicated. For example, as the Sun rises on the Moon, portions of spacecraft structures will be perpendicular to the rising Sun, and thus, will heat up more quickly than horizontal surfaces. The Surveyor III camera was elevated into the solar beam at sunrise and was not shaded during lunar daylight hours (Fig. 7A). Understanding how spacecraft surfaces heat and cool during daylight hours will greatly advance the LMS model predictions for microbial survival on and within complex spacecraft structures. Second, additional data are required on heat transfer processes into deeply embedded components of complex spacecraft structures during daylight hours. The warmer the internal high temperatures reach, the greater the biocidal effects will be observed due to the interactive effects of low pressure and elevated temperatures. And third, additional data are essential for defining the interactive effects of temperatures <100°C and low pressures close to cis-lunar vacuum conditions (10⁻⁸ to 10⁻¹⁰ Pa). As already discussed, the few articles found on interactive effects of vacuum and temperatures <100°C all indicated that warmer temperatures enhance the lethality of the vacuum. Thus, the LMS model predictions for deeply embedded spacecraft components might be increased if the deeply embedded components reach temperatures closer to 50–70°C. Biocidal effects are not limited to only temperatures >70°C (Figs. 2 and 3).

Kill kinetics for nonspore-forming and nonculturable microorganisms are required to constrain the LMS model regarding complex microbial communities and/or assemblages on spacecraft. Although most of the identified microbial contamination on lunar spacecraft appears to be in the domain Bacteria (e.g., Puleo et al., 1970, 1973a; Dillon et al., 1973) and some studies described a few fungal species on lunar spacecraft (Puleo et al., 1970, 1973a; Taylor et al., 1973), it appears that no more than ∼10% of the launched bioloads on lunar spacecraft are from the domain Eukarya. The LMS model focused here on the inactivation of B. subtilis spores, but data on other species, particularly species with higher resistance rates to the stresses modeled, would provide insights into refractory species with higher rates of survival than B. subtilis.

And lastly, the LMS model is based on a set of assumptions and empirical data in which a LD rate was defined as the interactive effects of all stressing agents culminating in a −10 log reduction in survivors. If new data indicate that the total microbial populations on launched lunar spacecraft were higher than modeled here, then the survival rates over time would be slightly longer. For example, if the estimated total probable bioburdens at lunar contact are estimated to be 2 logs higher than modeled here, increasing from 5.47 × 10⁸ to 5.47 × 10¹⁰ spores/cells per spacecraft (Table 1), the LMS model would still predict that microorganisms on most landed or crashed spacecraft on the Moon (except the Chang'e 3 and Yutu lander) would be inactivated by 2030 (Fig. 6 and Table 4). On average, increasing the total probable bioburdens at lunar contact by 1 log increases the time required to drop below one viable spore or cell by 50 lunations (Table 2) for the vacuum-only case on deep internal surfaces.

4.5. Future astrobiological targets for recovery of viable microorganisms on lunar hardware

Table 4 shows LMS predictions for bioburden levels for the last 10 spacecraft to have landed or crashed on the lunar surface up to January 1, 2019 (column 2) and January 1, 2030 (column 3). The LMS predictions for January 1, 2019 suggest that the last 10 lunar spacecraft may have residual viable microorganisms that are deeply embedded near the central cores of each spacecraft. For January 1, 2030 (a reasonable date for the return of humans to the Moon), 5 of the last 10 lunar spacecraft would be expected to harbor no viable microorganisms. Only the Chang'e/Yutu lander was estimated to plausibly harbor significant bioburdens near 1 × 10⁵ spores/cells on deeply embedded components (Table 4); a population of viable microbes that may be detected on returned spacecraft components. For all earlier spacecraft landed or crashed between September 14, 1959 (Luna 2) and August 18, 1976 (Luna 24) (Table 1), the LMS model predicts them to be free of viable Earth microorganisms.

The following high-priority astrobiology targets are proposed for collecting lunar archived payloads and equipment for microbial study.

First, although the most easily accessible materials on the Moon would be the surface experiments deployed by the Apollo astronauts (e.g., the Apollo 15 Heat Flow experiment) (Fig. 8A) or the Lunar Rover Vehicle (Fig. 8B), these payloads are of limited value for astrobiology studies because they are either small and poorly shielded (Fig. 8A) or are of similar unshielded construction as the Surveyor III lander (Fig. 7A). However, samples from these payloads would provide insights into how spacecraft materials have survived space weathering since their initial deployments, including the effects of rocket exhaust plumes during lander ascents (i.e., plumes can “sand-blast” spacecraft surfaces; see Immer et al., 2011).

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FIG. 8.

Apollo images depicting the complexity of the landed modules, equipment, rovers, and impact sites. (A) The maximum temperature observed for the Apollo 15 Heat Flow experiment was 100°C in the exposed surface cables (arrows). (B) The LRV was constructed of vented materials and compartments and was unpressurized. Thus, most surfaces exposed to solar thermal infrared would likely have reached temperatures given in Table 3 for the Surveyor I and III components. All internal surfaces of the LRV in thermal contact with the heated exterior would have been exposed to temperatures <140°C. (C) A nylon JETT bag with LM habitat refuse can be seen tossed underneath the Apollo 11 descent stage. (D) Impact site for the Apollo 13 Saturn IVB booster. Images were downloaded from the website (www.apolloarchive.com/apollo_gallery.html), except image D which was downloaded from (www.nasa.gov/mission_pages/LRO/multimedia/lroimages/lroc-20100322-apollo13booster.html). Images are courtesy of NASA: (A) NASA-JSC_AS15-92-12416HR, (B) NASA-JSC_AS17-134-20476, (C) NASA-JSC_AS11-4-5850, and (D) NASA-LCROSS_M109420042LE. LCROSS, Lunar Crater Observation and Sensing Satellite; LRO, Lunar Reconnaissance Orbiter; LRV, Lunar Rover Vehicle.

Second, the highest priority for an Apollo era microbial survey would be the nylon jettison (JETT) bags placed underneath the LM descent stages or tossed onto the lunar surface before leaving the Moon (e.g., Apollo 11 JETT bag) (Fig. 8C). The JETT bags contain disposable materials from within the crewed LM ascent stages before landing, including water containers, human waste, food wrappers, LiOH canisters (for airborne spores), and sleep restraints (dermal microbiota) (e.g., Anonymous, 1971, 1972). If the JETT bags with human waste and sleep restraints were placed under the LM descent stages, they would have the richest microbial bioburdens and species diversity of the accessible materials on the Moon (i.e., materials directly handled by human astronauts), and the bags have been mostly shielded from the worst of the thermal cycling conditions on the Moon. Furthermore, the LM descent stages are the largest intact structures on the Moon and would provide insights into shielding against ionizing radiation effects on biological systems.

Another interesting astrobiological target for recovering materials would be samples from one of the Apollo Saturn IVB booster or LM ascent stage impact craters (e.g., Fig. 8D). Assumption (A15) in the LMS model states that thermal heating of spacecraft during impact events was not incorporated because data were not available that would predict impact temperatures of such vehicles. Data on the thermal heating of impacted spacecraft and materials would advance the LMS model predictions for such events, and possibly significantly lower the survival rates of microorganisms for impacted spacecraft components.

Fourth, the spacecraft with the highest predicted surviving bioburden on the Moon is the Chang'e and Yutu lander (Table 4). The LMS model predicts that as of January 01, 2019, the Chang'e Yutu lander may retain ∼7.03 × 10⁷ viable spores/cells on deeply embedded surfaces. This population will be inactivated on average by only −0.02 logs per lunation (or −0.247 logs per year) by vacuum-only effects, and thus, by January 1, 2030, the abundance of viable microorganisms within the lander would be ∼1.29 × 10⁵ spores/cells. Of the spacecraft currently on the surface of the Moon, the Chang'e and Yutu lander may hold the greatest promise for the recovery of viable Earth microorganisms present on the Moon for at least 10 years.

And lastly, any new lunar spacecraft landed or crashed to the lunar surface during the next decade should be fitted with astrobiology payloads that could be recovered and analyzed for microbial survival, biosignature degradation, and materials alterations when humans return to the Moon around 2030. Such astrobiological experiments would require very little mass, but could greatly advance our understanding of how Earth microorganisms can withstand the perils of the lunar environment.