The goal of planetary protection as stated in NASA policy is prevention of forward and backward contamination (42
). This policy applies directly to the control of terrestrial organisms contaminating spacecraft intended to land, orbit, fly by, or be in the vicinity of extraterrestrial bodies. Viking mission landers were terminally heat sterilized to decrease the risk of forward contamination of Mars and to ensure that terrestrial microorganisms did not contaminate the life detection experiments (42
). However, the cost of designing and assembling the Viking landers was increased dramatically due to this requirement. In 1992 the Space Studies Board and the Committee on Space Research concluded from Viking mission data that Mars was less likely to support Earth-based life than previously thought (49a
). The non-life-detection Mars landing missions, such as Mars Exploration Rovers, did not require all rover components to be heat sterilized prior to launch. Instead, NASA relied on a series of sequential chemical and physical sanitation steps to maintain the cleanliness of the Mars Exploration Rovers (9
The question is whether forward contamination of Mars will be significantly decreased by the inherent harsh environment at the Martian surface. Spores of B. subtilis
have been shown to survive for up to 6 years under low-Earth-orbit conditions (15
). However, only shielding from UV radiation enabled B. subtilis
endospores to survive under these conditions for a long time (15
). The solar flux at the Martian surface is considerably less than that experienced in interplanetary space (15
), and there is the potential that atmospheric conditions could further attenuate UV irradiation (7
Previous UV resistance studies have utilized model dosimetric strains and indicated that the limit for survival of organisms is approximately 200 J m−2
). A recent study examined the survival of a laboratory strain, B. subtilis
HA 101, on spacecraft-qualified materials under simulated Martian UV irradiance conditions (46
). The results suggested that ~6 logs of spores exposed on spacecraft surfaces under the simulated UV conditions were inactivated within a few tens of minutes under Mars equatorial and clear-sky, 0.5-optical-depth conditions. Other researchers have examined a B. pumilus
strain that was isolated from a spacecraft assembly facility and reported that it maintained one of the highest levels of UV254
resistance reported for spores to date (28
). Since most of the previously published UV resistance information has been based on the use of laboratory strains, predictions of the actual survival and possible adaptation of terrestrial life on Mars are limited due to the lack of robust empirical data. The same lack of data could also hamper efforts to use UV irradiation as a sterilization method if the most resistant organisms are not tested during the creation of dose standards. For example, the current standard for UV disinfection of drinking water is 400 J m−2
. B. pumilus
SAFR-032 requires doses of 2,000 to 2,500 J m−2
, an order of magnitude greater than the standard, for complete sanitation (28
The current study is the first study to report the abilities of a wide range of Bacillus species recovered from spacecraft and associated facilities to survive simulated Martian UV exposure. The sources of the type strains used in this study for phylogenetic comparison to spacecraft-related isolates varied from soils in France to milk (Table ), reinforcing the widespread nature of spores on Earth. Correlations between type strain and spacecraft-related isolate resistance to UV irradiation were not observed. In some cases the type strain was more resistant to UV irradiation than spacecraft-related isolates (e.g., B. megaterium and B. cereus), but in general the spacecraft-related strains were more resistant to UV irradiation than the type strain of B. pumilus (Table ). The source of spacecraft-related isolates (air or surface) did not correlate with UV irradiation resistance (Table ).
Even though spacecraft assembly facilities are cleaned on a regular basis, it is evident that the resistance properties of spore-forming microbes allow them to adapt and persist in these environments. While it is difficult to speculate on the sources of spores within the spacecraft assembly facilities, soil is generally thought to be the main repository of spores in the environment (31
) and the most likely vector of entry in this case. Evidence of this was obtained from the discovery of a strain of B. mojavensis
in the JPL Mars Environmental Chamber Assembly Facility (Table ). The type strain of B. mojavensis
was isolated from a desert located within 100 miles of the JPL campus. B. pumilus
was the predominant microbe that was repeatedly recovered from spacecraft (Viking in 1972 to Mars Odyssey in 2001) and the assembly facility surfaces (JPL and KSC) (22
) that resisted various perturbations, including UV and gamma radiation, and H2
. There have been no reports of how this prevalent microbial species was transported into the facility or how the microbes adapted to survive in the conditions of the facility. The B. pumilus
strains tested in this study exhibited no noticeable phenotypic differences. Recent studies employing genetic fingerprinting grouped all JPL spacecraft assembly facility B. pumilus
isolates into three clusters (20
). Therefore, it is likely that B. pumilus
strains adapted over time to the conditions present in the spacecraft assembly facilities, and this may explain their elevated levels of resistance.
The data presented here indicate that spores of B. pumilus SAFR-032 are far more resistant to simulated Martian UV irradiation conditions than standard dosimetric strains are. Since B. pumilus SAFR-032 was isolated from a spacecraft assembly facility and exhibited enhanced UV resistance, it follows that any sanitation procedures involving UV irradiation should be based on the most UV-resistant microorganisms recovered from spacecraft. It is necessary to continue testing spacecraft contaminants in order to properly characterize the UV resistance of the viable bioload prior to launch.
Furthermore, during experiments in which spores of two different strains were mixed, it appeared that B. pumilus SAFR-032 spores protected the more UV-sensitive B. subtilis 168 spores. Specifically, colonies of B. subtilis 168 were not observed on plates following treatment with Martian UV irradiation for ~2 min or longer. However, when mixed with B. pumilus SAFR-032 spores, spores of B. subtilis 168 survived exposure to 5 or 10 min of Martian UV irradiation (Fig. ). Autoclaved B. pumilus SAFR-032 spores did not protect B. subtilis 168 spores, and the inactivation curve for spores of B. subtilis 168 was very similar to the curves generated for unmixed 168 spores at a density of 5 × 105 spores ml−1. Further research is necessary to elucidate the influence of viable B. pumilus SAFR-032 spores or spore components, such as spore coat proteins, on protection of UV-sensitive strains.
The spectral output of the JPL X-25 solar simulator used in this study was different than that of the KSC Martian UV simulator used by Schuerger et al. (46
), as shown in Fig. . Many factors can contribute to the spectral quality and output of UV irradiance lamps, including the age of the lamps, special coatings on the glass bulbs, and the chemical and physical composition of the bulbs. From the UV spectral irradiance at both JPL and KSC, it is evident that the KSC UV simulation had a higher UV flux at wavelengths less than 260 nm and was more lethal to both organisms tested. UV irradiation at wavelengths less than 260 nm has been shown to be highly lethal to microbes and coincides with the action spectrum of DNA, causing the most significant damage of the UV bandwidths (18
). Since the KSC simulator was richer in UVC than the JPL simulator, the difference in the inactivation rates supports the conclusion that UV irradiation in the 200- to 280-nm range is the UV irradiation that is most detrimental to spores. Therefore, any attenuation of UVC by dust or ice particles in the atmosphere may greatly enhance spore survival.
In summary, the results of this study suggest that the UV environment on Mars is extremely harsh and that most microorganisms exposed to the sun would be rapidly inactivated at equatorial latitudes. However, the existence of organisms like SAFR-032, whose survival was significantly greater than that of the standard lab strain, B. subtilis 168, should be considered when workers examine the biocidal nature of UV irradiation specifically on Mars with respect to future robotic or human exploration missions. In addition, further research is warranted (i) to determine the biocidal effects of low Martian pressure and extreme desiccation on the survival of bacteria protected from direct UV irradiation, (ii) to study the effects of Mars dust and nonbiological spacecraft residues (e.g., lubricants) on the survival of terrestrial microorganisms with Martian UV fluence rates, and (iii) to determine if low levels of diffuse UV irradiation permit adaptation of terrestrial microorganisms to different Martian conditions. The research described here demonstrated that the Mars UV environment is likely to be very detrimental to the survival of microbial species from Earth, but until the other questions mentioned above can be properly addressed, we must remain vigilant in processing spacecraft for Mars to reduce the possibility of forward contamination of landing sites.