The Mars Sample Return (MSR) mission architecture has perhaps the most stringent planetary protection (PP) protocols for Mars spacecraft since the Viking landers were heat sterilized prior to launch. The Perseverance rover is the first part of a three-spacecraft MSR program to return Martian rock and regolith samples by the mid 2030’s (Haltigin et al., 2022; Mattingly and May, 2011; Moeller et al., 2021). To prevent false positive in MSR samples by hitchhiking Earth microbes (i.e., making the round-trip voyage), the PP protocols for the Perseverance rover, Sample Retrieval Lander (SRL), and Earth Return Orbiter (ERO) must be more stringent than previous Mars landers or rovers. For example, the average bioburden at launch for the Perseverance rover wase 1.47 x 104 spores for the whole vehicle (Cooper et al., 2023) compared to 5.64 x 104 and 1.5 x 105 spores per vehicle for the Curiosity and InSight spacecraft, respectively (Benardini et al., 2014; Hendrickson, et al., 2020). In addition, the bioburden of the ACA on Perseverance was estimated to be 4.11 x 103 spores (Cooper et al., 2023); an unusually low number for Mars spacecraft subsystems that attests to the more stringent PP protocols used for sampling hardware for the MSR mission. In general, bacterial spores have been estimated to comprise between 10% (Dillon et al., 1973) and 40% (La Duc et al., 2007) of the total microbial bioburdens of spacecraft.
Deferring for the moment the discussion of the loss of viable bioburdens on MSR spacecraft during the Earth-to-Mars transit, microbial survival on the Martian surface will play a critical role in lowering the risk of false-positives in returned samples. The combined effects of 20 + biocidal factors on the Martian surface (see Rummel et al., 2014; Schuerger et al., 2013; Stoker et al., 2010) are likely to significantly reduce the viable bioburdens on spacecraft surfaces during the multi-year surface operations of the Perseverance and SRL vehicles. The biocidal conditions on the Martian surface are very likely to lower the risks of false positives from spacecraft components in MSR samples.
Although no quantitative and comprehensive Mars microbial survival (MMS) model currently exists, a preliminary MMS model was presented by Moores et al. (2007). The goal of the current study was to begin the development of a comprehensive MMS model by characterizing the inactivation rates of microbial bioburdens on and around the Adaptive Caching Assembly (ACA) of the Perseverance rover as a first-order estimate of microbial survival on MSR hardware involved in collecting samples for Earth return. Results support several overarching conclusions on the effects of solar UV irradiation on bioburden inactivation rates for Mars spacecraft.
First, data on the effects of UVC irradiation on microbial survival exist for a wide range of microorganisms recovered from spacecraft surfaces (Table 4 and references cited therein). When published UV-biocidal LD90 rates for common spacecraft microbiota are compared, the times required to achieve significant levels of inactivation are surprisingly fast for the Martian surface. For example, the average LD90 inactivation rate for the 20 bacteria listed in Table 4 was only 377 sec (i.e., 6.28 min) on upward facing horizontal surfaces on Mars spacecraft located close to the equator, at Ls 180, and under clear-sky conditions of tau = 0.5. This rapid inactivation rate continues for every log reduction thereafter as ‒1 log per 6.28 min until the inflection point of − 4 logs (approx. 25 min) is reached for UV-sensitive microbiota on spacecraft surfaces (Fig. 5; see Schuerger et al., 2019). The 2nd phase of biocidal activity in Fig. 5 is much slower and reflects UV inactivation refractory microorganisms that could be caused by multiple layers, partially UV-shielded, or genetically more UV-resistant spores.
Even for partially shielded locations on the upper decks of spacecraft, biocidal UVC photons can be reflected off other materials, and thus, biocidal UVC photons are likely to reach protected sites resulting in moderately fast rates of biocidal activity for all surfaces. For example, if a population of UV-resistant microbes similar to B. pumilus SAFR-032 requires only 0.83 sols to reach one SAL (Table 5), a protected site that receives only 10% of reflected UVC irradiation would only require approx. 8.3 sols to reach one SAL. Given that the lengths of most Mars rover missions are several years on the Martian terrain, achieving one SAL in only 8.3 sols for the upper decks of spacecraft is extremely fast.
Furthermore, the robotic arm of the Perseverance rover was elevated above the upper deck of the spacecraft for most of the initial few tens of sols on Mars. Thus, most surfaces that received direct and reflected UVC photons should reach one SAL within 8.3 sols (nominal case). In situations on the robotic arm or upper deck of Perseverance in which surface defects, pits, or overhangs exist (see Schuerger et al., 2005), solar UVC exposure times will be longer but not indefinite. As direct and diffuse UVC photons reach the bottoms of these surface features, a lethal dose of one SAL is likely to be achieved within a few tens of sols (Schuerger et al., 2005; 2006).
Second, the experimental setup used here (Fig. 1) yielded UVC reflectance values for analog Mars soils of approx. 1.3% at three different SZA’s. The results are lower than the approx. 5% UVC reflectance values for other Mars analogs described by Cloutis et al. (2008). Although experimental protocols differed between the current study and the work by Cloutis et al. (2008) that could explain the different results (e.g., hardware orientations between UV illuminators, targets, and detectors; a double-slit spectrometer used here versus a single-slit spectrometer used by Cloutis et al.; etc.), both studies are consistent by indicating that UVC reflectance of the terrain on Mars is likely to be on the low end of the scale and will likely not exceed 5% for most surface features.
Third, the data presented here confirms that UVC reflectance of spacecraft materials have a wide range of values (from < 1 to ~ 65%; e.g., Table 1) which can significantly increase the biocidal effects of UVC irradiation in partially shielded sites on spacecraft superstructures. The overall average for UVC of all spacecraft materials and SZA’s was approx. 10%. Thus, for partially shielded locations on upper deck components on Perseverance, multiple bounces of UVC irradiation off spacecraft components are likely to achieve one SAL levels of inactivity in relatively short time-steps between a few hours to a few sols (Table 5).
And fourth, the ACA system is located at the forward edge of the Perseverance rover and is comprised of complex 3-dimensional components and diverse surfaces (Fig. 7) that are likely to offer multiple sites for partially shielded locations. Thus, we modeled biocidal inactivation rates for the ACA as either (i) surfaces that directly face downward, or (ii) components that require an additional single secondary UVC bounces off of adjacent spacecraft materials.
The best predictor of accumulating a single lethal dose (i.e., one SAL) for downward facing surfaces is the Geometric Shielding Ratio (GSR) described by Moores et al. (2007). The GSR captures the effects of both the height off the terrain of a rover or lander (i.e., the z-axis of the undersides above the terrain) and the x- and y-axes of the rover underbellies. The GSR for the Perseverance rover is approx. 2.8, which yields a prediction of one SAL of ~ 93 sols for the ACA contaminated with UVC-resistant spore-forming bacteria similar to B. pumilus SAFR-032 (Table 5). Subsequently, if a second UVC bounce is required for a partially protected site within the ACA material‒and assuming an average UVC reflectance of 10% for spacecraft materials (Tables 1, 2, and 3)‒one SAL might require up to 930 sols on UV-shielded surfaces within the ACA.
The bioburden reduction models in Fig. 5 were used to make specific predictions in Table 5 on the numbers of sols to achieve bioburden reductions of up to one SAL for several scenarios on the Perseverance rover. Although rapid bioburden reductions are likely for UV-sensitive microbiota on spacecraft (1st phase of the UV biocidal kill curves; Fig. 5; see Schuerger et al., 2006), one SAL is defined as a bioburden reduction of ‒12 logs (Craven et al., 2021). Thus, even if lower than normal bioburdens persist on spacecraft hardware than are typical (i.e., the ACA bioburden of 4.11 x 103 spores; Cooper et al., 2023), one SAL will still require the time and UVC flux to achieve ‒12 logs bioburden reductions derived from larger populations of spores in empirical lab tests. The explanation for this counter intuitive statement is the possibility that the lower bioburden of the ACA described by Cooper et al. (2023) might be composed of a refractory population of spores. Thus, the times to achieve one SAL given in Table 5 should be used for all bioburden levels achieved during pre-launch PP cleaning protocols.