Introduction
Since the discovery of Martian meteorites (Wasson & Wetherill Reference Wasson, Wetherill and Gehrels1979; Becker & Pepin Reference Becker and Pepin1984; Dreibus & Wänke Reference Dreibus and Wänke1984, Reference Dreibus and Wänke1985) it is a generally accepted supposition that rock fragments can escape from planetary bodies, e.g. ejected from very large impact craters, and that interplanetary transfer of matter has occurred several times during the history of our Solar System (O'Keefe & Ahrens Reference O'Keefe and Ahrens1986; Vickery & Melosh Reference Vickery and Melosh1987). However, it is still an open question, whether living matter has been transported between the planets of our Solar System by the same mechanism, and, if so, whether resistant organisms can withstand the severe strain of a journey through the Solar System. During such a hypothetical interplanetary transfer, the organisms would have to cope with the following three major challenges: (1) the escape process, (2) the long-duration exposure to space and (3) the capture and entering process. Although it will be difficult to prove that resistant organisms could survive this cascade of strenuous attacks, estimates of the chances of the different steps of the process to occur can be obtained from measurements in space and laboratory simulation experiments, and from model calculations (Mileikowsky et al. Reference Mileikowsky, Cucinotta, Wilson, Gladman, Horneck, Lindegren, Melosh, Rickman, Valtonen and Zheng2000; Clark Reference Clark2001; Horneck et al. Reference Horneck2008, Reference Horneck, Klaus and Mancinelli2010; Nicholson Reference Nicholson2009; Onofri et al. Reference Onofri2012).
In the SPORES (Spores in artificial meteorites) experiment of the EXPOSE-R (Exposure facility attached to the URM-D of the Zvezda Module of the ISS) mission on board of the International Space Station (ISS), we have addressed the question of the chances and limits of life to be transported from one body of our Solar System to another by natural processes by testing experimentally step 2, i.e. whether the meteorite material offers enough protection against the harsh environment of space for spores to survive a long-term stay in space. For this purpose, spores of the bacterium Bacillus subtilis 168, which have proven their high resistance to outer space conditions in several previous space experiments (reviewed in Horneck et al. Reference Horneck, Klaus and Mancinelli2010) were studied under the following conditions:
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• Snapshot experiment: The first objective was to produce a snapshot of a hypothetical journey of spores located at the outer layer of a meteorite. For this purpose, a thin layer of spores was mixed with different concentrations of simulated Martian regolith powder (Mars regolith analog, MRS07) and exposed the selected parameters of outer space under the conditions, given by the ISS, i.e. free access to space vacuum, minimal protection against solar and cosmic radiation, no special orientation to the sun.
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• Protection experiment: The second objective was to determine the protective effects of meteorite material against the different parameters of space, applied individually or in selected combinations, especially space vacuum, cosmic radiation and full spectrum of solar extraterrestrial ultraviolet (UV) radiation. For this purpose, spore layers with and without MRS07 were exposed in stacks of three layers to outer space parameters, where the top layers provided a certain UV-screening to the subjacent layers.
The SPORES experiment of the EXPOSE-R mission was part of a set of experiments performed by the ROSE consortium to study the Response of Organisms to Space Environment (Rabbow et al. Reference Rabbow2014).
Material and methods
Bacterial strain and sporulation procedure
The experiment SPORES was carried out with endospores of B. subtilis wild-type strain 168 (DSM 402) obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig , Germany. Spores were produced by cultivation in the liquid Schaeffer sporulation medium as described in Nicholson & Setlow (Reference Nicholson, Setlow, Harwood and Cuttings1990) and Moeller et al. (Reference Moeller, Horneck, Rettberg, Mollenkopf, Stackebrandt and Nicholson2006). In brief, an inoculum was grown in the Schaeffer sporulation medium at 37 °C under vigorous aeration and the level of sporulation was daily examined by light microscopy. At a sporulation rate of 99%, which was reached after 3–4 days, the spores were harvested by centrifugation, purified and stored in distilled water at 4 °C. The spores gained by this procedure were free of vegetative cells, germinating spores, and other cell debris, as determined by the phase contrast microscopy.
Sample preparation
Snapshot experiment
Twenty microlitres of a suspension of spores of B. subtilis 168 containing 1×107 colony-forming units were placed aseptically on the surface of sterile quartz discs of 7 mm in diameter (Herasil 102). Part of the spore suspension was mixed with 0.01 g ml−1 sterile simulated Mars regolith (MRS07) powder composed of 47.7 wt% Montmorillonite, 9.9 wt% Kaolinit and 21.3 wt% hematite (19.17 wt% Fe2O3, 1.3 wt% SiO2 and 13.0 wt% Anhydrit) (Museum für Naturkunde, Humboldt Universität, Berlin). The aqueous spore suspension or spore/dust mixture was dried overnight under laboratory conditions at (22±2)°C and (33±4)% relative humidity and then stored in the dark until further use in the experiment. In this experiment, the spores were arrayed in monolayers.
For scanning electron microscopy (SEM), some extra samples with and without MRS07 were sputtered with a gold film of approx. 10 nm and examined with a Zeiss field-emission SEM DSM 982 Gemini with an acceleration voltage of 5 kV using the Everhart–Thornley SE-detector and the inlens SE-detector, as described in Moeller et al. (Reference Moeller, Rohde and Reitz2010) (Fig. 1). As shown clearly in Fig. 1(B), in samples mixed with MRS07 only few spores were uncovered (<20% of the spores were free-lying), whereas the majority of the spores was entrapped in aggregates of the artificial Martian regolith.
Fig. 1. Scanning electron micrograph images of artificial Martian regolith (MRS07; a); wild-type B. subtilis 168 spores (Snapshot experimental setup: 107 spores on 7 mm diameter quartz disc, white arrowheads indicate uncovered spores) covered with MRS07 (b) and uncovered spores (c).
Protection experiment
Fifty microlitres of the spore suspension containing 3×108 colony-forming units – either in pure aqueous suspension or mixed with 0.01 or 0.03 g ml−1 MRS07 powder – were placed on the surface of MgF2 discs (11.9 mm in diameter, 1 mm in height) and dried overnight as in the Snapshot experiment. These layers were about 2–3 spores thick. The spore-laden discs were accommodated as stacks of three inside a metal sleeve. They were separated by an O-ring from each other and fixed by use of the space proved glue (Wacker Silikone) (Fig. 2). The attenuation of UV irradiance by the overlying sample layers on MgF2 discs was spectroscopically determined (Hitachi U-3200 double monochromator spectrophotometer). Table 1 shows the reduction of the transmission at 200 nm<λ>400 nm of spare samples that were prepared as the flight samples.
Fig. 2. Scheme of the sample stack of three MgF2 discs loaded with a dried layer of Bacillus subtilis 168 spores (Protection experiment).
Table 1. Protection experiment of SPORES: reduction of the transmission by spore-laden MgF2 disc
For both types of experiments two identical experimental setups were prepared to be used either in the flight experiment or in the mission ground reference (MGR) experiment performed in the Planetary and Space Simulation facilities (PSI) at the DLR (Rabbow et al. Reference Rabbow2014).
Experimental setup
The samples were integrated into the compartments of the three trays of the EXPOSE-R facility (Fig. 3) according to an accommodation plan (Rabbow et al. Reference Rabbow2014). For each test parameter, three replicates were used. The exposure conditions (Ar atmosphere at 1023 hPa or space vacuum; solar irradiation at λ>110 or >200 nm; full solar irradiance or attenuation by 2 or 4 orders of magnitude) are listed in Table 2.
Fig. 3. Part of EXPOSE-R during spaceflight attached to the URM-D platform of the Zvezda module of the ISS (credit NASA).
Table 2. Experimental parameters of the SPORES experiment on board of EXPOSE-R and fluences of solar spectral ranges on top of the windows and at the samples sites, as calculated by RedShift (the difference in fluences for samples under identical optical filters but located at different positions of the EXPOSE-R platform are due to a gradient in the insolation of the platform caused by shadowing and orientation of the ISS)
In the Snapshot experiment, beneath the UV-exposed upper layer of spore-loaded discs, there were either one or two layers of samples kept in the dark. They experienced the same environmental conditions as the upper sample layer, except the insolation. In the Protect experimental part, a dark sample layer was located beneath the sample stacks. An identical arrangement of samples was produced in the ground control trays that served for the MGR (Rabbow et al. Reference Rabbow2014). In addition, laboratory controls were prepared at the same time and from the same batch of spores as the flight and MGR samples, and they were stored in the dark under ambient laboratory conditions (temperature 20±2 °C and relative humidity 33±5%).
Flight protocol
The EXPOSE-R facility with its samples was launched to the ISS November 26th, 2008, it was attached by extravehicular activity (EVA) to the universal work-place outside platform of the Zvezda module of the ISS (URM-D) located outside of the Russian Service Module Zvezda on March 11, 2009, and it returned to Earth on March 9, 2011 after exposure duration in outer space of 681 days. Data of general health status of EXPOSE-R, temperature, insolation and galactic cosmic radiation were regularly sent via telemetry to the ground. Owing to a breakdown of the onboard computer about 42% of the measured environmental data was lost and a total mission environmental profile could not be obtained. Therefore, the UV fluence at the sample site (Table 2) was calculated by RedShift Design and Engineering BVBA, Belgium, from the available ISS mission flight data (Rabbow et al. Reference Rabbow2014). Post-flight inspection of the retrieved EXPOSE-R facility revealed that the windows of the vented compartments, i.e. those that were open to space vacuum through a valve, were tinged with a brownish colour. Spectroscopic analyses provided evidence of organic residues on the windows. It is still debated, whether this discolouring was caused by photolysis of the samples (Demets et al. Reference Demets2014) or by condensation of combustion products from the visiting Soyuz and Progress spacecrafts (W. Seboldt, personal communication). The SPORES samples were then returned to DLR for analyses.
Mission ground reference
The purpose of the MGR was to mimic the environmental conditions of the EXPOSE-R flight mission as closely as possible on ground in the PSI at the DLR in Cologne. In the MGR, a similar set of trays and sample arrangement was used as in the EXPOSE-R flight model. The samples were exposed to simulated environmental conditions according to the flight data, with respect to vacuum, temperature oscillations (restricted by technical limits) and UV200–400 nm radiation. These flight data of temperature, UV exposure and general health status of EXPOSE-R were obtained by telemetry or – in the cases of data loss due to computer failure – by model calculations. The MGR started on December14th, 2009 and ended on October 17th, 2011. The mission parameters were simulated under the limits of the Earth-bound possibilities as closely as possible and are described elsewhere in detail (Rabbow et al. Reference Rabbow2014).
Sample analysis
Spore survival
The spores were recovered from the quartz or MgF2 discs by applying the polyvinyl alcohol (PVA)-stripping method as described in Horneck et al. (Reference Horneck, Rettberg, Reitz, Wehner, Eschweiler, Strauch, Panitz, Starke and Baumstark-Khan2001). In brief, the dry spore layers were covered with a sterile 10% aqueous PVA solution (30 μl for the 7 mm discs; 50 μl for the 11.9 mm discs). After drying, the PVA layer with the enclosed spores was stripped off the discs with sterile forceps. This procedure was repeated three times for every sample to ensure complete retrieval of the spore layers. Subsequently, these three-pooled spore-PVA layers were resuspended in 1 ml sterile aqua demineralized. This procedure does not affect spore viability and it resulted in approximately 95% recovery of the spores as demonstrated in Horneck et al. (Reference Horneck, Rettberg, Reitz, Wehner, Eschweiler, Strauch, Panitz, Starke and Baumstark-Khan2001). After appropriate dilution of the spore suspension in 10−1 dilution steps their ability to form macroscopic visible colonies on nutrient agar plates was determined after incubation for 24 h at 37 °C. The surviving fraction was determined from the quotient N/N 0, with N is the number of colony-forming units of the flight or MGR sample and N 0 that of the initial colony formers at the time of flight/MGR sample preparation.
Numerical and statistical analyses
The results of the spore survivability were compared statistically using Student's t test. Values were analysed in multigroup pairwise combinations, and differences with P values of ⩽0.05 were considered to be statistically significant.
Photoproduct analysis
In addition to the survival of the spores, the UV-induced DNA damage was quantified by high-performance liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (HPLC–ESI–MS/MS). For this, spores of samples of the top layers of the Protection experiment and samples of the ROSE 4 experiment (Rabbow et al. Reference Rabbow2014) of identical exposure conditions were pooled and the spore DNA was isolated as described in Moeller et al. (Reference Moeller, Douki, Cadet, Stackebrandt, Nicholson, Rettberg, Reitz and Horneck2007). The photoproduct analysis was performed by HPLC–ESI–MS/MS after enzymatic digestion of the DNA to detect dimeric photoproducts from adjacent pyrimidines released as modified dinucleoside monophosphates (Douki et al. Reference Douki, Court, Sauvaigo, Odin and Cadet2000). With this technique it is possible to detect simultaneously the cis-syn and trans-syn cyclobutane thymine dimers (c-s T<>T and t-s T<>T, respectively), the related pyrimidine (6–4) pyrimidone adduct (6–4 TT), and its Dewar valence isomer (Dewar TT) as well as the corresponding thymine–cytosine dimeric photoproduct, CT and cytosine dimeric photoproduct (CC) bipyrimidine photoproducts (BPP) in addition to the spore-specific photoproduct 5,6-dihydro-5(α-thyminyl)thymine (SP) (Moeller et al. Reference Moeller, Douki, Cadet, Stackebrandt, Nicholson, Rettberg, Reitz and Horneck2007). The results are given as DNA BPP per 106 bases.
Results
Snapshot experiment
Spores in monolayers, simultaneously exposed to space vacuum and the complete spectrum of solar extraterrestrial radiation (λ>110 nm) at a UV fluence F 110–400 nm=(8.0±1.4)×105 kJ m−2 during the 681 days lasting EXPOSE-R space mission were completely inactivated (N/N 0<10−7) (Table 3). During the analyses after the mission none of the spores were able to germinate and to form a colony. Neither replacing space vacuum by an Ar atmosphere nor mixing the spores with MSR07 powder altered those results (Table 3). They confirm earlier observations on the extreme lethality of extraterrestrial solar UV radiation (Nicholson et al. Reference Nicholson, Munakata, Horneck, Melosh and Setlow2000; Horneck et al. Reference Horneck, Klaus and Mancinelli2010, Reference Horneck, Moeller, Cadet, Douki, Mancinelli, Nicholson, Panitz, Rabbow, Rettberg, Spry, Stackebrandt, Vaishampayan and Venkateswaran2012). Attenuation of the UV fluence by about 4 orders of magnitude to 51±10 kJ m−2 for spores in Ar atmosphere resulted in few colony formers [N/N 0=(1.0±0.1)×10−6], but not for spores in space vacuum at a UV fluence of 65±13 kJ m−2. If the spores in Ar were mixed with MRS07 powder, their survival was further increased to [N/N 0=(7.2±0.7)×10−6]; however, for spores UV irradiated in space vacuum, mixing with MRS07 powder did not result in any survivors.
Table 3. Survival of spores of Bacillus subtilis 168 in the Snapshot experiment of SPORES after spaceflight during the EXPOSE-R mission
One asterisk (*) indicates the spore survivability, determined after UV irradiation at λ>110 nm (either in argon atmosphere or vacuum), which was significantly different from the respective survival values obtained from spore UV irradiation at λ>200 nm (Student's t test; P⩽0.05).
Two asterisks (**) indicate the survival value of (non-UV-irradiated) spores covered with MRS07 regolith that was significantly different from the corresponding survival value of uncovered spores (Student's t test; P⩽0.05).
Survival of spores was slightly increased, if the extraterrestrial UV radiation was cut-off at 200 nm (Table 3), although the UV fluence was only slightly reduced compared to that at λ>110 nm (Table 2). Survival rates reached the 10−6 and even 10−5 ranges, if the UV radiation was attenuated by 4 orders of magnitude and MRS07 powder was mixed to the spores (Table 3). These results demonstrate that the vacuum UV radiation (V-UV) range (λ<200 nm) was highly efficient in inactivating the spores. Statistical analysis showed that spores were significantly more sensitive to UV at λ>110 nm than to UV at λ>200 nm, in a dose-effect-dependent manner (Table 3).
Spores kept in the dark but experiencing all other environmental conditions as the sun-exposed spores during the 681 days spaceflight showed much higher survival rates: Mostly in the 10−3 ranges, if mixed with MRS07, or in the 10−4 ranges, if without any admixture (Table 3). For comparison, the laboratory controls, that were prepared together with the flight samples but stored in the laboratory during the whole mission of EXPOSE-R, showed a very high survival rate of [N/N 0=(8.7±0.9)×10−1]. Furthermore, it can be shown that dark sample spores covered with MRS07 showed significantly higher survival rates compared with those obtained from uncovered spores indicating the protective attributes of MRS07 with regard to extreme desiccation (Tables 3–6).
A comparable low survival rate – if any – was found for spores exposed in the laboratory to the simulated EXPOSE-R space flight conditions during the MGR, where the solar simulator provided a spectrum of λ>200 nm and the vacuum amounted to a pressure of 1.7×10−3 Pa (Table 4). Spores in monolayers without any admixture did not survive this UV irradiation at a fluence of 8.5×105 kJ m−2. Attenuation of the UV irradiance by 4 orders of magnitude gave survival rates in the 10−6 range for spores without any admixture, and in the 10−5 range for spores, mixed with MRS07 powder. The dark controls that were located beneath the irradiated samples survived with rates in the 10−3 range, if they were exposed without any admixture, and in the 10−2 range, if they were mixed with MRS07 powder and kept in Ar atmosphere.
Table 4. Survival of spores of Bacillus subtilis 168 in the Snapshot experiment of SPORES after the EXPOSE-R MGR in the PSI at the DLR
Two asterisks (**) indicate the survival value of (non-UV-irradiated) spores covered with MRS07 regolith that was significantly different from the corresponding survival value of uncovered spores (Student's t test; P⩽0.05).
Protection experiment
In the Protection experiment, all samples were kept in space vacuum and arranged in stacks of three to mimic different depths of a meteorite (Fig. 2). The MgF2 windows were transparent for the full solar irradiance at λ>110 nm. Whereas spores in the top layer showed a similar low survival in the 10−7–10−6 ranges as the spores of the Snapshot experiment, partial protection from harmful solar extraterrestrial UV radiation was reached with increasing depth. In the bottom layer of stacks composed of three layers spore-laden MgF2 discs, a survival rate in the 10−4 range [N/N 0=(3.8±1.0)×10−4] was reached, if the spore layer was mixed with high concentrations of MRS07 powder (Table 5). The presence of MGR07 powder in high concentration led also to a higher survival rate of the dark controls, which amounted to 23% survivors (Table 5).
Table 5. Survival of spores of Bacillus subtilis 168 in stacked layers of the Protection experiment of SPORES after spaceflight during the EXPOSE-R mission
One asterisk (*) indicates spore survival values of (UV-irradiated) spores covered in MRS07 regolith that was significantly different from the survival values obtained for uncovered spores (Student's t test; P⩽0.05).
Two asterisks (**) indicate survival value of (non-UV-irradiated) spores covered with MRS07 regolith that was significantly different from the corresponding survival value of uncovered spores (Student's t test; P⩽0.05).
Spores in laboratory simulations of MGR showed a similar tendency as the space samples: High inactivation in the 10−7–10−6 survival ranges of spores in the top layer, with increasing survival rates up to the 10−3 range with increasing depth; high survival rates of the dark controls, with MRS07 mixture as well as without admixture (Table 6).
Table 6. Survival of spores of Bacillus subtilis 168 in stacked layers of the Protection experiment of SPORES after the EXPOSE-R MGR in the PSI at the DLR
One asterisk (*) indicates survival values of (UV-irradiated) spores covered with MRS07 regolith that was significantly different from the survival values obtained for uncovered spores after the same treatment (Student's t test; P⩽0.05).
Photoproduct formation
The photoproduct analysis by HPLC–ESI–MS/MS requires sufficient amount of pure DNA extracted from the treated spores. Owing to the low samples size of UV-irradiated spores it was not possible to extract pure DNA in an amount that was sufficient for the photoproduct analysis. For comparison, we present here the not yet published photoproduct analysis by HPLC–MS/MS of B. subtilis 168 spores that were flown with the Exposure facility attached to the balcony of the European module Columbus of the ISS (EXPOSE-E) mission, where a larger number of exposed spores were available. The environmental conditions (UV range and fluence at sample site with 100% transmission of 500–600 MJ m−2, cosmic radiation dose, space vacuum and temperature range) were quite similar to those of the EXPOSE-R mission (Horneck et al. 2012). The analyses identified SP as the only photoproduct found in the DNA of the spores, exposed to outer space in low Earth orbit (LEO) as well as to simulated Martian climate conditions (Table 7). In the space experiment, there was a reverse relationship between UV fluence and amount of SP detected: The higher the UV fluence, the lower was the frequency of SP. This was for spores exposed to outer space, as well as for those kept under simulated Martian climate. Under the latter conditions with UV cut-off at 200 nm, spores exhibited more SP than under full solar UV irradiation at λ>110 nm.
Table 7. Spore photoproduct measured in the DNA of Bacillus subtilis 168 spores that were exposed to outer space conditions and to simulated Martian conditions during the 1.5 years lasting EXPOSE-E mission and data from the corresponding MGR
a 1.6% AR, 0.15% O2 and 2.7% N 2 in CO2 atmosphere.
A similar reverse relationship between UV fluence and amount of SP was found for spores of the MGR experiment that were exposed to simulated outer space, but with a UV cut-off at 200 nm for all test conditions (Table 7). The fraction of SP was even higher than in the space flight experiment, although the UV fluences of the MGR were slightly lower and no UV below 200 nm reached the spores. Only the simulated Martian conditions of the MGR gave a lower amount of SP at a 1000 times lower UV fluence.
Discussion
SPORES of the EXPOSE-R mission onboard of the ISS has provided further experimental data to the discussion on the likelihood of Lithopanspermia, i.e. the natural transfer of microorganisms from one planet to another via impact-ejected rocks (Mileikowsky et al. Reference Mileikowsky, Cucinotta, Wilson, Gladman, Horneck, Lindegren, Melosh, Rickman, Valtonen and Zheng2000; Clark Reference Clark2001; Nicholson Reference Nicholson2009; Onofri et al. Reference Onofri2012; Scalzi et al. Reference Scalzi, Selbmann, Zucconi, Rabbow, Horneck, Albertano and Onofri2012). Travel times up to millions of years have been estimated from cosmic radiation exposure of Martian meteorites (Eugster et al. Reference Eugster, Herzog, Marti, Caffee, Lauretta and McSween2006); however, model calculations indicate also shorter travel times (Gladman et al. Reference Gladman, Burns, Duncan, Lee and Levison1996). During this interplanetary journey, microorganisms have to cope with a complex interplay of strenuous conditions: Solar extraterrestrial UV radiation, Galactic cosmic radiation, solar particle events, space vacuum and temperature extremes. Although the SPORES experiment mimics only a 2 years snapshot of such a hypothetical journey between the planets, its results provide insights into the limiting conditions for Lithopanspermia.
Extraterrestrial Solar UV radiation
The SPORES results confirm earlier observations on the extreme lethality of extraterrestrial Solar UV radiation (reviewed in Nicholson et al. Reference Nicholson, Munakata, Horneck, Melosh and Setlow2000; Horneck et al. Reference Horneck, Klaus and Mancinelli2010). Not any survivors were found after 2 years exposure of monolayers of B. subtilis spores to the full spectrum of extraterrestrial solar UV (Table 3). Exposure of bacterial spores to the full UV spectrum (λ>110 nm) that included V-UV (λ<200 nm) was more deleterious to the spores than exposure to simulated Mars UV radiation (λ>200 nm) as was also shown by Wassmann et al. (Reference Wassmann2012). The admixture of MRS07 powder, used as Martian meteorite analogue, did not remarkably increase their survival rate. DNA injury is conjectured as main cause of spore inactivation, and the so-called spore photoproduct (SP) was the only pyrimidine base damage detected in spores exposed during the EXPOSE-E mission to similar conditions as during EXPOSE-R (Table 7). This is the first time that the analysis of photoproducts in the DNA of space-exposed spores has been presented. Unexpectedly, a lower level of SP was determined in samples exposed to the highest UV fluences. In contrast to cyclobutane thymine dimers that undergo photoreversion or to 6–4 TTs which can be converted into their Dewar valence isomer, no photochemical process is known for SP that can explain this observation. It may be proposed that SP is reversed into unmodified thymine as the result of radical processes triggered by the high-energy UV photons (λ<200 nm), which are known to induce DNA ionization (Melvin et al. Reference Melvin, Cunniffe, O'Neill, Parker and Roldan-Arjona1998). This could involve the radical reaction mediated by the SP lyase. Further experiments are however required to challenge this hypothesis. The results confirm that spores in monolayers residing at the outside of a rock travelling through interplanetary space will be efficiently inactivated within very short time by extraterrestrial solar UV radiation.
Galactic cosmic radiation
Galactic cosmic radiation penetrates to greater depths of a rock and may reach spores in the rock's interior, where the cells would be shielded from solar UV radiation. During the 2 years lasting SPORES experiment, the radiation doses were too low to affect a significant number of cells: there was no remarkable difference between survival of dark space and dark MGR spores (Tables 3–6). Only the sparse hits by heavy ions of cosmic radiation may lead to inactivation of spores. Detection of those effects needs localization of the particle's trajectory in the spore layer, as successfully demonstrated in the Biostack experiments, flown on the Apollo missions (Facius et al. Reference Facius, Bücker, Hildebrand, Horneck, Höltz, Reitz, Schäfer and Toth1978; reviewed in Horneck et al. Reference Horneck, Klaus and Mancinelli2010). These heavy ions of cosmic radiation may finally be a threat to spores inside rocks during extended travel times in space (Mileikowsky et al. Reference Mileikowsky, Cucinotta, Wilson, Gladman, Horneck, Lindegren, Melosh, Rickman, Valtonen and Zheng2000).
Space vacuum
There was no significant difference in the survival of spores kept in the dark, either in space vacuum at about 10−4 Pa or in Ar at about 105 Pa. This resistance of B. subtilis 168 spores against space vacuum is well documented (reviewed in Horneck et al. Reference Horneck, Klaus and Mancinelli2010) and one of the strategies of bacterial spores to survive extreme arid conditions. Additional protection against desiccation caused by space vacuum can be provided by the presence of chemical protectants (Horneck et al. Reference Horneck, Bücker and Reitz1994) or by simulated Martian regolith, as demonstrated in this SPORES experiment (Tables 3–6).
Temperature extremes
During the EXPOSE-R mission, the temperature varied between −(24.7±2.0)°C and +(49.5±2.0)°C, depending on the orbital cycles and position of the ISS. It is not clear how far these temperature fluctuations influenced the survival of the spores, because the same temperature profile was simulated in the MGR. However, the survival rate of space and MGR samples that were kept in Ar at 105 Pa was lower by 2–3 orders of magnitude than that of laboratory controls that were kept for 2 years in the dark at ambient pressure and room temperature in parallel to the EXPOSE-R mission.
Conclusions
The SPORES experiment of the EXPOSE-R mission has again demonstrated the limits for the Lithopanspermia scenario. The chances to survive inside of impact-ejected rocks during an interplanetary journey requires: (i) that the microorganisms are endolithic, i.e. reside in the interior of rocks; (ii) that they are capable of long-term dormancy with increased resistance to desiccation, radiation and extreme temperature fluctuations; (iii) that they are capable of repair of the generated DNA damage after termination of the space travel.
Acknowledgements
The authors would like to thank the European Space Agency (ESA) for the flight opportunity and the support by the ESA EXPOSE-R team, Kayser-Threde GmbH, München, for their technical support, RedShift for the solar irradiance calculations, and the team at DLR MUSC for the analysis of the flight data, the assembling of the flight experiment and the performance of the MGR test. The authors are very grateful to Manfred Rohde for his generous support with the SEM analysis of the spore samples. We thank Simon Barczyk, Maria Bohmeier and Andre Parpart at the DLR, Institute of Aerospace Medicine, for technical assistance during preparation and analysis of the experiment. Part of these investigations has been carried out in the frame of the project ‘Spores’ and ‘ELO’, code 50WB 0528 and 50WB 0830, supported by the German Bundesministerium für Witschaft und Energie.
Author disclosure Statement
No competing financial interest exists.
