Synergistic interactions among multiple space biocidal conditions provide more accurate predictions on the lethality of the interplanetary environment than single-factor experiments. However, multifactorial assays are often difficult to complete due to rapidly increasing combinations of factors as independent variables. For example, if three factors are considered for an assay, the total possible combinations are six (i.e., 3! = 6 combinations). Four factors quickly escalates to 24 combinations (i.e., 4! = 24). Furthermore, selecting the dominant factors to test often leads to significant knowledge gaps for more subtle interactions. In the current study, the interactive effects of high vacuum (VAC), simulated solar heating (HEAT), and simulated solar UV irradiation (UV) were examine in combination to examine the potential lethality of interplanetary space on three bacteria commonly recovered from spacecraft surfaces.
Vacuum alone was found to have no significant effect on the survival of B. atrophaeus, B. pumilus, or B. subtilis over the course the short-term exposures tested here compared to the lab controls maintained at a sea level pressure of 1 bar (P > 0.05) (Figs. 3, 4, 5, 6). In contrast, longer-term exposures have reported loss of viability of up to ‒0.5 to ‒2 logs between 6 and 69 months for B. subtilis maintained as dried monolayers on metal coupons (see Fig. 1 and references in Schuerger et al. [18]). All subsequent interactive assays described below were conducted under a background pressure of 0.05 (Fig. 3) to 10− 6 (Figs. 4, 5, and 6) mbar maintained for 48 hrs. We found no evidence in the current experiments, or in the literature, that the difference between 0.05 and 10− 6 mbar affected the results described herein.
In contrast, in all VAC + HEAT assays (Figs. 3 and 6), the presence of vacuum increased the lethality of high-temperature (i.e., 100°C tested here) by as much as ‒7 logs for B. pumilus (Fig. 3B) and approx. ‒4 to ‒5 logs for B. atrophaeus and B. subtilis. Thus, the synergism between VAC + HEAT had a significantly greater effect on the UV-resistant strain of B. pumilus SAFR-032 as compared to the other two Bacillus spp. Results are consistent with other studies in which vacuum increased the lethality of high-temperatures at 59–60°C by ‒0.5 logs [29, 30, 31] and at 100°C by ‒6 logs [18]. In one interesting study, the synergistic effects of vacuum applied during long-term heating assays against the heat-tolerant Bacillus sp. ATCC 29669 boosted the overall lethality of high temperatures between 125 and 170°C by several to many orders of magnitude under vacuum [32]. However, synergism appeared to abate when vacuum and lab-air samples were exposed to 200°C [32]. And finally, incubating B. subtilis spores at ‒193°C (80 K) increased survival significantly compared to room temperature of 20°C when both were under vacuum [29]. These data support the conclusion that the lethality of diverse temperatures when spores are under vacuum increases compared to similar tests under lab pressures closer to 1013 mbar.
The inactivation kinetics of HEAT alone at 1 bar, or VAC + HEAT, followed linear models as presented in Fig. 3 here or elsewhere [18, 29, 30, 32] suggesting that viable outliers did not persist beyond the linear extensions of the models when samples were exposed to high temperatures under vacuum.
Synergism was also observed between VAC + low-UV treatments (Fig. 4; UV flux at 3.16 AU within the asteroid belt) and between VAC + high-UV treatments (Fig. 5; UV flux at 1.36 AU close to perihelion for Mars) exhibiting as much as ‒5 to ‒7 logs of increased lethality for all three Bacillus spp. at 1, 5, and 10 min of UV exposure. In general, the inactivation kinetics followed linear models with the exceptions of VAC + high-UV for B. pumilus (Fig. 5B) and B. subtilis (Fig. 5C). The results were consistent with a sizeable body of literature depicting synergism between UV irradiation and vacuum tested against Bacillus spp. (e.g., [33, 34, 35, 36, 37, 38, 39]. Synergism between VAC + UV has also been reported for non-spore forming bacteria Escherichia coli [40, 41]and Deinococcus radiodurans [41]. The level of increased lethality was typically several orders of magnitude in these studies depending on the lengths of exposures to the combined VAC + UV conditions.
When all three space factors were combined into one assay (Exp-4), the overall bioburden reductions reached approx. ‒8 logs (Fig. 6) after only brief exposures to HEAT (i.e., only 1 h at 100°C) and UV (i.e., only 2 min during the heat-pulse) in a VAC background of 10− 6 mbar for 48 hrs. Figure 2B depicts the overall timing of the three factors. Longer times for either HEAT or UV yielded 100% inactivation for all test samples during the 3-factor experiments (data not shown); meaning, that no detectable viable spores were recovered. Exp-4 (i.e., VAC + HEAT + UV) required sublethal doses of each parameter to test for synergism. To measure synergistic interactions with predictive knowledge on how each factor contributed to the overall biocidal effects, only experiments with sublethal dosages can be run. For example, if a 24-h concomitant VAC + HEAT + UV simulation were conducted, how can one resolve the contribution of each factor when results yield zero survivors?
Linear models were chosen here to represent the inactivation kinetics for combinations of VAC, HEAT, and UV exposures. In most cases, the linear models fit the data best. However, two exceptions are noteworthy. For spores of B. pumilus (Fig. 5B) and B. subtilis (Fig. 5C), very precipitous inactivation was observed for the first high-UV exposures up to approx. 5–10 min followed by a tailing effect due to single-digit surviving ‘outliers’ at longer time-steps (Table S4). Schuerger et al. [25] reported similar effects of tailing when 7 Bacillus spp. were exposed to a simulated Mars surface UVC flux, but eventually they were able to sterilize aluminum coupons after longer exposures. Tailing is generally not observed for heat-sterilization of microbes but can be observed with UV-sterilization processes due to subtle shading effects of spores entrapped in pits, cracks, other surface defects, or present as multi-layered spore aggregates [22]. Redline linear models in Figs. 5B and 5C are suggested plots for the 1st phase of the inactivation plots for B. pumilus and B. subtilis, respectively. The redline models are more realistic for the biological sensitivity of both species to UV irradiation. The dash-dot lines for both species were derived for the full datasets with all single-digit outliers included.
Synergism works to greatly enhance the lethality of the space environment on spacecraft bioburdens. The results presented herein focused on interactive effects of three space conditions including vacuum, simulated solar heating, and simulated solar UV irradiation. Results were consistent across all assays and synergism was observed in all multi-factor experiments. In addition, these results are consistent with a large body of literature; some-of-which are cited above. In addition, synergism has been described between HEAT + UV exposures for the fungus Aspergillus nidulans [42]. Interestingly, one study on the interactions between vacuum and ionizing radiation (IRAD) demonstrated a sort of a “positive synergistic interaction’ in which spores of Bacillus megaterium, B. subtilis var. niger, Clostridium sprorgenes, and Aspergillus niger were found to survive better under γ-radiation when exposed under high vacuum of approx. 10− 9 mbar versus lab air [43]. Presumably, the γ-rays ionized the gas molecules in the lab air forming O‒, O2‒, and/or NOx‒ radicals which contributed to the biocidal conditions within the sealed glass vessels.
The results presented here can be used to estimate the Sterility Assurance Levels (SALs) for spacecraft surfaces exposed to combination of VAC, HEAT, and UV during interplanetary transits. The medical equipment industry standard approach [26, 27, 28] to estimating SALs are to estimate the times required to inactivate 1 x 10− 6 spores by a specific sterilization protocol and then doubling those exposure times (i.e., thus achieving 10− 12 bioburden reductions; syn. here of ‒12 logs). Table 1 presents SAL estimates for all linear models in Figs. 3, 4, and 5. The SALs in Table 1 were estimated by extending the linear models to ‒12 logs and are similar to following the industry standard protocol outlined above.
SAL estimates for the slowest combination of VAC + low-UV (Table 4) approached 60–70 min of exposure for sun-facing surfaces. The fastest times to one SALs were observed for the redline linear plots in Figs. 5B and 5C at 13.1 and 17.6 min for B. pumilus and B. subtilis, respectively. Overall, these inactivation kinetics are in agreement with survival rates given for the Lunar Microbial Survival (LMS) and Cruise Phase Microbial Survival (CPMS) models [18, 17]. In the LMS, when all factors were combined into a single prediction per lunation (14.77 d of solar illumination in each 29.53 d lunation) for Lunar spacecraft, as many as ‒2479 logs of bioburden reduction were possible. The value equates to an accumulation of 207 SALs (i.e., [‒2479 logs] ÷ [‒12 logs per SAL]) for external spacecraft surfaces on the Moon per lunation. Thus, single SALs are expected to occur over extremely short periods of time within the inner Solar System (Table 1; [17]), and extremely high numbers of accumulated SALs can be achieved over a few weeks when spacecraft surfaces are exposed to vacuum, solar heating up to at least 100°C, and solar UV irradiation.
Results suggest that the VEEGA trajectory for the Europa Clipper (EC) spacecraft will keep the EC spacecraft within the inner solar system for up to 3.5 years (Buffington, 2014) potentially accumulating in excess of 1.7 x 104 SALs (i.e., 207 SALs for every 14.77 d of solar illumination on the Moon in the LMS model multiplied by 3.5 years) during the exposures to cis-Lunar/Earth conditions or higher. In addition, even after leaving the 1–2 AU environment, the EC spacecraft’s external surfaces will continue to accumulate SAL exposures throughout the mission increasing confidence that launched bioburdens from Earth are unlikely to impact the mission science.
Regarding the MSR mission architecture, the best orbital location in which vacuum, solar heating, and solar UV will impact microbial survival would be during operations in low-Mars-orbit (LMO) for the Orbital Sample (OS) holding device and the external surfaces of the ERO spacecraft. However, no quantitative model has yet been developed to precisely model the LMO, ERO, or OS environments in regard to how quickly each phase of the MSR mission will accumulate adequate SAL values for mitigating both forward and back contamination.