Survival of Bacillus pumilus Spores for a Prolonged Period of Time in Real Space Conditions

Abstract

To prevent forward contamination and maintain the scientific integrity of future life-detection missions, it is important to characterize and attempt to eliminate terrestrial microorganisms associated with exploratory spacecraft and landing vehicles. Among the organisms isolated from spacecraft-associated surfaces, spores of Bacillus pumilus SAFR-032 exhibited unusually high resistance to decontamination techniques such as UV radiation and peroxide treatment. Subsequently, B. pumilus SAFR-032 was flown to the International Space Station (ISS) and exposed to a variety of space conditions via the European Technology Exposure Facility (EuTEF). After 18 months of exposure in the EXPOSE facility of the European Space Agency (ESA) on EuTEF under dark space conditions, SAFR-032 spores showed 10–40% survivability, whereas a survival rate of 85–100% was observed when these spores were kept aboard the ISS under dark simulated martian atmospheric conditions. In contrast, when UV (>110 nm) was applied on SAFR-032 spores for the same time period and under the same conditions used in EXPOSE, a ∼7-log reduction in viability was observed. A parallel experiment was conducted on Earth with identical samples under simulated space conditions. Spores exposed to ground simulations showed less of a reduction in viability when compared with the “real space” exposed spores (∼3-log reduction in viability for “UV-Mars,” and ∼4-log reduction in viability for “UV-Space”). A comparative proteomics analysis indicated that proteins conferring resistant traits (superoxide dismutase) were present in higher concentration in space-exposed spores when compared to controls. Also, the first-generation cells and spores derived from space-exposed samples exhibited elevated UVC resistance when compared with their ground control counterparts. The data generated are important for calculating the probability and mechanisms of microbial survival in space conditions and assessing microbial contaminants as risks for forward contamination and in situ life detection. Key Words: Bacillus pumilus—Spores—Space conditions—International Space Station—Mars atmosphere—UV radiation. Astrobiology 12, 487–497.

1. Introduction

T he Committee on Space Research (COSPAR) planetary protection policy establishes guidelines for spacefaring nations “to avoid organic-constituent and biological contamination in space exploration” (COSPAR, 2002, 2011). For this purpose, it is necessary to enumerate aerobic spore-forming bacteria on spacecraft surfaces as a proxy for measuring the cleanliness of spacecraft intended to land, detect signatures of life, or return planetary samples to Earth. It has consistently been found that spore formers constituted ∼10% of the cultivable microorganisms isolated from surfaces of various spacecraft (Puleo et al., 1977; La Duc et al., 2003; Ghosh et al., 2009). This may be due to the methods employed to assess the microbial burden of these surfaces, as these findings were heavily influenced by heat-shock procedures that optimized the detection of spores while excluding most vegetative cells. The spore-forming microbes are of particular concern in the context of planetary protection because their endospores are highly resistant to various physical and chemical conditions. These conditions include ionizing and UV radiation, desiccation, and oxidative stress. The wild-type endospores isolated from spacecraft and associated surfaces have been found to be more resistant to these conditions compared to laboratory strains (La Duc et al., 2004; Link et al., 2004), as well as sterilization procedures (La Duc et al., 2003, 2007), and the harsh environment of outer space or simulated planetary surfaces (Nicholson et al., 2000). In microbial diversity surveys, researchers have isolated several microbial species that inhabit various spacecraft assembly facilities despite the stringent cleaning and maintenance protocols in place (La Duc et al., 2003, 2004, 2007; Newcombe et al., 2005). When 45 spacecraft surface–associated Bacillus spore lines were screened, 19 isolates showed high resistance to UVC (254 nm) irradiation after exposure to 1000 J/m² (Newcombe et al., 2005; La Duc et al., 2007). It was found that spores of Bacillus species isolated from spacecraft surfaces were more resistant to the martian UV radiation climate than the standard laboratory strain B. subtilis 168 (Link et al., 2004). These observations support the hypothesis that the special conditions of ultraclean spacecraft building and assembly facilities (low nutrient level, controlled humidity, filtered air circulation to maintain low particle concentration, controlled temperature, and decontamination measures) select for the most resistant organisms (Venkateswaran et al., 2001).

Among the organisms isolated from spacecraft-associated habitats, spores of B. pumilus SAFR-032 exhibited unusually high resistance to simulated martian UV when compared with the other strains of the same species (Link et al., 2004; Kempf et al., 2005). With regard to the D value (the dose required to reduce the number of surviving microorganisms by one log), the spores of B. pumilus SAFR-032 are 6 times more resistant to UV irradiation and 50 times more resistant to gamma irradiation than B. subtilis spores (Newcombe et al., 2005). To better understand the basis for this elevated resistance, the genome of the SAFR-032 strain was sequenced and compared with other Bacillus genomes (Gioia et al., 2007). The SAFR-032 genome paradoxically lacks several genes present in related Bacillus species responsible for UV or H₂O₂ resistance but includes other genes that have not been found in related species. At this point, it is not known whether SAFR-032 spores derive their elevated resistance properties from intrinsic protective mechanisms, elevated repair capabilities (during germination), or a combination of the two. Nevertheless, microorganisms that have the capacity to withstand extreme environmental conditions are of concern to planetary protection efforts because they may escape sterilization protocols and possibly withstand the hostile environment of interplanetary space, which would thereby allow them to contaminate other Solar System bodies (Kempf et al., 2005).

Hence, in this context it was important to investigate in situ the resistance of B. pumilus SAFR-032 to relevant extraterrestrial environments. The European Space Agency (ESA) EXPOSE-E facility, attached to the International Space Station (ISS) as part of the European Technology Exposure Facility (EuTEF), was used as a test bed for various space conditions. EuTEF consisted of five individual exobiology experiments, including PROTECT, which investigated the resistance of spores to the open space environment. Survival indices calculated after the spores were exposed to UV radiation might help explain how shielding affects spore survival. Such information will prove invaluable for future risk assessments of forward contamination (Rummel, 1989), in addition to furthering our general understanding of the potential of microbial survival and proliferation on the surface or shallow subsurface of Mars.

Reported here are the findings of survival of spore-forming bacteria isolated from spacecraft-associated environments exposed under real space conditions for a prolonged period of time (∼18 months). Also tested was the hypothesis that conditions resulting in reduced cellular water content (such as vacuum and extreme desiccation) are major predisposing selective factors for both microbial persistence in interplanetary space and potential subsequent proliferation on the martian surface. One of the prevailing opinions with regard to radiation-resistance mechanisms is expression of proteins responsible for these traits. To further understand the resistance mechanisms, a viable strategy is to identify the highly expressed proteins. Such studies have been performed earlier for non-spore-forming radiation-resistant bacteria (Narumi et al., 2004). The exact resistance mechanisms and protein machinery responsible for resistance traits observed in SAFR-032 are still unknown. In addition, the elevated resistance properties of first-generation spores or cells generated from the space-surviving SAFR-032 strain were tested. Studies on the physiological and proteomic changes of space survivors might reveal the selective adaptation and microbial persistence of spacecraft-associated microbes in interplanetary space and subsequent proliferation on the martian surface.

2. Materials and Methods

2.1. Bacterial strains and purification of spores

Bacillus pumilus SAFR-032 was isolated from the Jet Propulsion Laboratory–spacecraft assembly facility (JPL-SAF) Building 179 (Kempf et al., 2005). For sporulation and purification, B. pumilus SAFR-032 strain was initially grown on tryptic soy agar (TSA) and incubated at 32°C for 24 h. A nutrient broth sporulation medium (NSM) was used to induce sporulation, and spores were harvested and purified as previously described (Schaeffer et al., 1965; Nicholson and Setlow, 1990). Briefly, a single purified colony was inoculated into NSM, and after 3 days of growth at 32°C, cultures were examined via microscopy to determine the level of sporulation. Once the number of free spores in the culture was ∼99%, the culture was harvested and spores were purified. Purified spores were resuspended in sterile deionized water, heat-shocked (80°C for 15 min), and stored in glass tubes at 4°C.

The identity of all B. pumilus samples (control and returned from space) was confirmed with 16S rDNA sequencing analysis. Bacterial small subunit rRNA genes of purified strains were polymerase chain reaction (PCR) amplified with B27F and B1492R primers, and PCR conditions were followed as described elsewhere (Ruimy et al., 1994). After purifying with QIAquick columns (Qiagen), 16S rDNA amplicons were fully bi-directionally sequenced. The identification and phylogenetic relationships of tested strains were determined by comparison of individual 16S rDNA sequences to sequences in the public database (http://www.ncbi.nlm.nih.gov).

2.2. Spacecraft-qualified aluminum coupon preparation

High-grade aluminum (Al 6061-T6) currently being considered for use in in situ and sample return hardware was selected for this study, based on the recommendation of the JPL Spacecraft Assembly Engineering Group. Mill–finish mirror-polished aluminum (alloying elements, in percent: 0.65 Si, 0.44 Fe, 0.27 Cu, 0.02 Mn, 0.96 Mg, 0.20 Cr, 0.02 Ti) was cut into 13 mm diameter “coupons.” Care was taken to avoid scratching the material's surface, and coupons were visibly inspected and rejected if they were scratched or did not have clean edges. Conventional autoclaving to ensure sterility of the metal was not attempted since the surface property of the aluminum metal was shown to be altered due to excessive heating (Venkateswaran et al., 2004). Nevertheless, the cleaning process employed in this study rendered the metal coupons essentially sterile. Coupons were pre-cleaned with clean room–grade polyester wipes (Coventry 6209 c-prime, Freon-washed), saturated with acetone to remove residual adhesive, degreased with Freon-vapor for 1 h, and finally rinsed with isopropyl alcohol and dried in filtered air at room temperature (Jet Propulsion Laboratory, 1990; Venkateswaran et al., 2004). The sterility of the metal coupons was randomly assayed by directly placing the cleaned coupons into tryptic soy broth media and checking for absence of turbidity after incubating at 25°C for 48 h.

2.3. Spore seeding of the coupons

Prior to use, materials and equipment needed for sample preparation and analysis were cleaned to sterility according to standard microbiological protocols. For the preparation of the biological test samples, standard methods developed for previous space-exposure experiments were applied (Horneck et al., 1984, 1994). The density of each spore inoculum was enumerated by serial dilution spread plate assays. Each pre-cleaned coupon was seeded with 100 μL of spore suspension, which was pre-calculated to contain a defined spore number between 10⁷ and 10⁸ total spores. The spore-seeded aluminum coupons (Fig. 1) were dried at room temperature overnight and subsequently stored in sterile screw-capped 15 mL Falcon centrifuge tubes. The tubes with the spore-seeded coupons were numbered and mailed to the German Aerospace Center (DLR) for integration into the EXPOSE-E facility.

FIG. 1.

Electron micrographs of Bacillus pumilus SAFR-032 spores on aluminum coupons before and after exposure to space conditions. Arrows in “control before flight” panel indicate presence of spores under uneven surfaces of the coupon.

2.4. Spore recovery from coupons

Spores were recovered from aluminum coupons by the application and peeling off of polyvinyl alcohol (PVA) films (Tauscher et al., 2006). One hundred microliters of sterile 10% PVA, prepared in water, was applied in a thin layer over the spores on each coupon. After drying in an incubator for 1 h at 37°C, the flexible PVA film that contained embedded spores was peeled off the coupon with a sterile scalpel and forceps and placed into a glass test tube. This process was performed twice on each sample coupon for quantitative spore recovery. The peeled films for each sample were pooled and dissolved completely in 2 mL of sterile deionized water, and the spores were resuspended by vortex-mixing. Serial 10-fold dilutions of the spores were prepared in sterile phosphate-buffered saline (PBS) (up to 10⁻⁶), and 100 μL of spore suspension from each dilution was evenly spread on TSA plates in duplicate. One hundred microliters of sterile PBS was spread on TSA plates in duplicate as a negative control. All the TSA plates were incubated at 32°C for 24 h. Witness plates were kept open throughout the experiment in the laminar hood to assess contamination during the experimental procedure.

2.5. Selection of UV₂₅₄ resistance

Purified spores of B. pumilus SAFR-032 (control and space-returned strains) were diluted in PBS (pH 7.2) to a density of 10⁶ per milliliter. Initial spore density was estimated by serial dilution plating before each exposure. A low-pressure handheld mercury arc-UV lamp (UVP, Inc.; model UVG-11; UVC, 254 nm) was placed at a fixed height over the sample, and the UV flux at the surface of the spore suspension was measured with a UVX digital radiometer (UVP, Inc.). Exposure times necessary to yield fluences from 200 to 4000 J m⁻² at the sample surface were determined (UV flux was 1 J m⁻² s⁻²). Under aseptic conditions, each spore suspension was placed within a biohood, in an uncovered 50 mm glass Petri dish, and stirred with a magnetic stir bar (3–5 mm in length) while being exposed to UV irradiation. When 10 mL (10⁶/mL) of spore suspension was added to the 50 mm Petri dish, the height of the liquid was 3 mm. The liquid spore suspension was stirred gently (100 rotations per minute) to avoid splashing; this setup allowed the spores to be exposed to UVC evenly. From these, sample volumes of 100 μL were removed at specific time points, serially diluted, and spread atop TSA or R2A agar (Difco catalog no. 218263) plates.

2.6. Experiment hardware of the PROTECT experiment on board the ISS

The ESA EXPOSE-E facility, which accommodated the PROTECT experiment biological samples, was launched on STS-122 Columbus on 7 February 2008. On 15 February 2008, EXPOSE-E, as part of the EuTEF platform, was mounted to the outside of the Columbus module, to expose diverse samples to various space conditions. The EuTEF infrastructure was accommodated on the Columbus External Payload Adaptor, which functioned as an automated module for several technology payloads (seven scientific and two technical instruments), and consisted of 18 different experimental setups. The tray-like structures were built for exposure to a wide range of space environments. EXPOSE-E was decommissioned on 1 September 2009, retrieved on 2 September 2009, and returned to Earth on 12 September 2009 with STS-128 Discovery.

EXPOSE-E consisted of three flight hardware trays (see Horneck et al., 2012; Rabbow et al., 2012). Each tray consisted of four compartments, each accommodating 16 sample stacks beneath an optical filter window. Each stack was composed of six biological layers; only the top layer was exposed to solar electromagnetic radiation, and the remaining five layers were kept in darkness. As part of the PROTECT experiment, 16 sample stacks were assigned to B. pumilus SAFR-032. The spore count for top-layer coupons in each exposure condition was about 10⁷ spores/coupon, while the middle (second from the top layer) and bottom (third from the top layer) coupons had about 10⁸ spores/coupon. Additional coupons were prepared with both spore densities and stored at room temperature to serve as controls. These samples, along with space-returned samples, were eventually analyzed for spore survival.

During the 18-month mission, the samples were exposed to a range of conditions, including space vacuum (10⁻⁷ to 10⁻⁴ Pa); galactic cosmic radiation (130–190 mGy); and either the full spectrum of solar extraterrestrial electromagnetic radiation (λ>110 nm) with fluences of (8.0–8.8)×10⁵ J/m² (below a 0.1% transmission neutral density filter) and (5.5–6.1)×10⁸ J/m² (100% transmission insolated samples), or they were exposed to a simulated martian UV radiation climate (λ>200 nm) with fluences of (5.3–5.7)×10⁵ J/m² (below a 0.1% transmission neutral density filter) and (4.0–4.3)×10⁸ J/m² (100% transmission insolated samples). All fluences were calculated for the biologically active UV wavelength region 200–400 nm. Temperature varied between −20°C and +59.6°C, depending on the orientation of the ISS to the Sun. More information about the physical characteristics of this mission are reported elsewhere (Rabbow et al., 2012). The specific exposure conditions of the PROTECT samples were as follows:

(1) UV-Space: In the orbit of the ISS, the full spectrum of extraterrestrial solar electromagnetic radiation is reached at an intensity of 1 solar constant. Since it is known that solar UV radiation is the most damaging part of the space environment, different fluences were achieved by using optical neutral density (magnesium fluoride) filters that allow a UV range of >110 nm to pass through. In addition, this “UV-Space” setup experienced the total flux of galactic cosmic radiation. The UV-Space tray was vented to space vacuum. In the vicinity of the ISS, the pressure is about 10⁻⁴ Pa (Horneck et al., 1988).

(2) UV-Mars: Suprasil quartz filters placed in the top window allowed UV radiation of wavelengths >200 nm to pass through. This kind of radiation profile is similar to that expected at the surface of Mars (Patel et al., 2003). The Mars atmosphere in the flight hardware was generated by filling it with 1.6% argon, 0.15% oxygen, and 2.7% nitrogen in carbon dioxide. This portion of the EXPOSE-E tray was sealed, and the pressure of the flight hardware system at the beginning of the mission was set at 600 to 1000 Pa (6–10 mbar). This “UV-Mars” setup simulated the exposure of spores outside a robotic Mars lander when arriving at and residing on the surface of Mars.

(3) Dark-Space: When shielded against the solar UV radiation mentioned above, the condition of the EXPOSE-E tray would simulate the exposure of spores inside a robotic satellite during flight. However, one has to consider that during interplanetary flight the pressure will likely be lower by several orders of magnitude compared with low-Earth orbit. This “Dark Space” setup was achieved by the vented compartment in the dark area.

(4) Dark-Mars: The UV-shielded part in the “Mars tray” was called “Dark-Mars.” The Mars atmosphere in the flight hardware was generated by filling it with 1.6% argon, 0.15% oxygen, and 2.7% nitrogen in carbon dioxide. The pressure of the flight hardware system at the beginning of the mission was 600–1000 Pa.

2.7. Mission ground reference conditions

The mission ground reference (MGR) provided a control experiment that was performed in space simulation facilities at the DLR with flight-identical hardware and samples. Data received from the mission—in particular, the environmental data on temperature oscillations, and UV radiation profiles—were simulated as closely as possible. Performed in parallel to the flight mission, the identical MGR experiments and samples were exposed to vacuum and UV radiation modulated to mimic those on the ISS for the entire mission duration. For EXPOSE-E, two ground trays similar to those in space, which were equipped with identical samples of the PROTECT flight experiments, were evacuated or filled with simulated martian atmosphere at martian pressure. The whole MGR was performed continuously for 1.5 years. The MGR solar simulator created UV radiation similar to space by emitting wavelengths >200 nm; the biologically active wavelength region was 200–400 nm.

2.8. Microscopy

An Olympus (Napa, California) phase-contrast microscope (BX-60) was used to determine the refractile nature of the spores. A field-emission environmental scanning electron microscope (FE-SEM) (Philips XL30, FEI Co., Potomac, Maryland) was used for nondestructive examination of spores (see below). Specimen preparation procedures, which often lead to sample artifacts, are not necessary when using the FE-SEM.

A FE-SEM provides high spatial resolution combined with low electron beam accelerating voltage. The low-beam voltage of the FE-SEM allows examination of electrical insulators without having to deposit a surface-conducting (carbon or metal) layer to eliminate specimen charging, which can lead to a distorted and often completely unusable image. The deposition of a conducting material to control charging can complicate the analysis. In many situations, a low electron beam voltage intrinsically results in a much sharper image, especially of thin structures composed of elements of low atomic number. A Philips (FEI, Hillsboro, Oregon) FE-SEM (XL-50) was used to analyze a majority of the samples. Elemental analysis is possible in a scanning electron microscope equipped with an energy-dispersive X-ray (EDX) analyzer. EDX is based on the analysis of the characteristic X-rays emitted when an electron beam is incident on a sample. Unfortunately, the spatial resolution obtainable with EDX is at best about 1 μm. The acceleration voltage for analyzing aluminum samples was ∼10–20 kV. In the high-vacuum mode, secondary electron images were acquired for both metals. Similar settings were maintained when different models or scanning electron microscope instruments were used.

2.9. Proteomic analyses of “space-return” B. pumilus spores

Difference gel electrophoresis (DiGE) is a modification of 2D polyacrylamide gel electrophoresis. Two or three separate protein samples can be labeled with different fluorescent dyes prior to separation, which enables accurate analysis of differences in protein abundance between samples. The 2D DiGE proteomic analyses protocols, as previously published, were employed in this study (Yan et al., 2002). Briefly, the metal coupons with B. pumilus spores exposed to all four space conditions, and a ground control sample, were placed in suitable buffer (10 mM Tris-HCL, 5 mM magnesium acetate, pH 8.0), and crude proteins were extracted. This step was repeated three times to maximize the protein yield from the coupon. The pooled crude protein extracts were further lysed by using 2D cell lysis buffer (30 mM Tris-HCL, pH 8.8, containing 7 M urea, 2 M thiourea, 4% CHAPS, and 1% lithium dodecyl sulfate). Protein concentrations were determined by protein assay and adjusted to the desired concentration. Equal amounts of protein extract from paired samples (e.g., control, UV-Space, and Dark-Space) were labeled by CyDye DIGE fluors (size and charge matched), and the spectrally resolvable dyes enabled simultaneous co-separation and analysis of samples on a single multiplexed gel (Yan et al., 2002). Up to three samples were simultaneously separated on a single 2D gel by using isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. In the study, control (unexposed), UV-Space, and Dark-Space spore samples were simultaneously analyzed. Likewise, the Mars sample sets were paired in the same experiment. After electrophoresis, the gels were scanned with a Typhoon image scanner. Each scan revealed one of the CyDye signals (Cy2, Cy3, or Cy5). ImageQuant array image analysis software was used to generate the image presentation data, including the single and overlay images. The images were then subjected to DeCyder software analysis. DeCyder in-gel analysis software automatically located and analyzed multiplexed samples in the same gel. It also enabled complex analysis of multiple gels and provided comparative analysis and accurate measurement of differential protein expression. The DeCyder software was used to identify a design for spot picking; then the Ettan Spot Picker was employed to automatically pick protein spots of interest from the 2D gel. Finally, protein identification with mass spectrometry was carried out.

3. Results

3.1. Morphology of “space-surviving” spores

The surface properties of the aluminum metal coupons and the purity, as well as the monolayer configuration of spores exposed to various space and simulated martian conditions, are shown in Fig. 1. Even though the aluminum coupons are mirror polished (ASTM 2002), the FE-SEM micrographs revealed the presence of pits and faults. These locations are potential spots for spores to hide such that the UV rays would be rendered ineffective at penetrating the micrometer-layer–thick aluminum flakes and the biological specimens would ultimately be protected. The B. pumilus SAFR-032 spores prepared exhibited monolayers when 10⁷ spores were seeded per 13 mm diameter coupons (Fig. 1). The control spores before and after 1.5 years of exposure to space conditions did not show any deformation in their morphology. Although fewer spores were visible in the 1.5-year UV-Space–exposed specimen, the morphology of the remaining intact spores was not altered (Fig. 1).

3.2. Survival assessment of spores exposed to space conditions

Prior to flight experiments, ∼1.1×10⁷ SAFR-032 spores were seeded onto individual coupons, and ∼43% of these spores were recovered from the ground laboratory control after 1.5 years of storage as dry layers in the laboratory on Earth (Horneck et al., 2012). The reduction in laboratory control spores may have been due to the 1.5 years of aging and desiccation. Survival of B. pumilus SAFR-032 spores after exposure to space conditions is shown in Fig. 2. Solar extraterrestrial electromagnetic radiation (UV-Space, Top, Fig. 2A) and simulated martian conditions with 100% UV transmission (UV-Mars, Top, Fig. 2A) killed almost all spores seeded. However, from four coupons, 19 strains were isolated, and cells of the first generation of these “space survivors” were harvested and appropriately stored in cryobeads in 10 replicates. All experiments in which “space survivors” were used were carried out with these first-generation cells. The solar extraterrestrial electromagnetic radiation with 0.1% transmission neutral density filter (UV-Space, Top, Fig. 2B) reduced ∼2-log of the spores, whereas the 0.1% transmission alone eliminated only one log when exposed to UV-Mars conditions (Fig. 2B). Unlike real space-exposed conditions, the simulated MGR conditions for both UV-Space and UV-Mars did not dramatically reduce the survival of SAFR-032 spores. The reduction in spores was ∼3-log for UV-Space-MGR and ∼2-log for UV-Mars-MGR conditions (Top layers, Fig. 3A). However, survival of SAFR-032 spores was more or less similar for Earth (Fig. 3B) and real space sample sets (Fig. 2B) under solar extraterrestrial electromagnetic radiation with 0.1% transmission neutral density filter.

FIG. 2.

Survival of Bacillus pumilus SAFR-032 spores after exposure to space conditions. (A) Solar extraterrestrial electromagnetic radiation with 100% transmission of insolated samples. (B) Solar extraterrestrial electromagnetic radiation with 0.1% transmission neutral density filter. All fluences were calculated for the biologically active UV wavelength region 200–400 nm. Data points represent the averages and standard errors of the mean of duplicate determinations from each of two separate experiments. The top layer was exposed to solar electromagnetic radiation. The spore count for top-layer coupons in each exposure condition was about 10⁷ spores/coupon, while the middle (second from the top layer) and bottom (third from the top layer) coupons had about 10⁸ spores/coupon. Details of filters, UV fluences, simulated martian conditions are given in Materials and Methods. CFU, colony-forming units.

FIG. 3.

Survival of Bacillus pumilus SAFR-032 spores under MGR conditions. (A) Simulated solar extraterrestrial electromagnetic radiation with 100% transmission of insolated samples. (B) Simulated solar extraterrestrial electromagnetic radiation with 0.1% transmission neutral density filter. All fluences were calculated for the biologically active UV wavelength region 200–400 nm. Data points represent the averages and standard errors of the mean of duplicate determinations from each of two separate experiments. Details of filters, UV fluences, simulated martian conditions are given in Materials and Methods.

The spore samples placed just beneath the top layers were not exposed to solar extraterrestrial electromagnetic radiation (middle and bottom layers, Fig. 2); they did not show any dramatic reduction in the survival of SAFR-032 spores. Furthermore, B. pumilus SAFR-032 spores that were kept in the dark in the EXPOSE facility showed 10–40% survival rate even after 18 months' exposure to real space conditions (100%, or 0.1% solar radiation transmission compartment). In contrast, when the spores were kept in the dark in simulated martian atmospheric conditions in EXPOSE on board the ISS (Fig. 2, all layers), the survival rate increased to 40–100%. Similar observations were made when the spore samples were exposed to MGR conditions (Fig. 3).

3.3. Enhanced UVC resistance of “space-surviving” strains (spores and vegetative cells)

Spores purified from the first-generation vegetative cells arising from all four sets of exposed coupons and the one set of ground control coupons (five strains each per condition) were further screened for their UVC (254 nm) resistance (0–2000 J/m²). A majority of the spores perished after 1000 J/m² UVC (data not shown). Spores that exhibited equal or greater survival compared with the ground control set (one each) were further quantitatively measured for their enhanced survival against UVC radiation (Fig. 4). The spores retrieved from UV-Space (56T-2) and UV-Mars (183T-1) showed resistance to UVC at 2500 J/m² (4-log reduction) in comparison with the Dark-Space (40T-5) and Dark-Mars (168T-5) spores. In addition, the spores of UV-Space (56T-2) exhibited highest UVC resistance with significant numbers of survivors observed at cumulative dosages up to 4000 J/m² (Fig. 4). The first-generation vegetative cells arising from these four SAFR-032 “space-surviving” variants and the ground control SAFR-032 strain were then exposed to UVC conditions from 0–900 J/m². As expected, the vegetative cells of the ground control SAFR-032 strain did not withstand even 200 J/m² UVC. All “space-surviving” variants showed resistance of up to 500 J/m² UVC (∼3-log reduction, Fig. 5). Unexpectedly, the Dark-Space SAFR-032 variant (40T-5 strain) persisted until 900 J/m² UVC (∼4-log reduction, Fig. 5).

FIG. 4.

UVC resistance of “space-surviving” Bacillus pumilus SAFR-032 spores. After Earth return, spores of surviving populations from each condition were purified from first generation and exposed to various cumulative doses of UVC (254 nm). Details of experimental conditions are given in Materials and Methods.

FIG. 5.

UVC resistance of “space-surviving” Bacillus pumilus SAFR-032 vegetative cells. After Earth return, first generation of vegetative cells isolated from each condition were exposed to various cumulative doses of UVC (254 nm). Details of experimental conditions are given in Materials and Methods.

3.4. Proteomic characterization of “space-exposed” B. pumilus spores

The proteins extracted directly from five metal coupons, which were either “space exposed” to four different conditions or ground control spores, were analyzed. The extracted reaction mixtures contained proteins from both dead and viable spores. Comparative analyses based on 2D-DiGE proteomic characterization of space-returned B. pumilus SAFR-032 spores that were exposed to UV-Space and UV-Mars are shown in Fig. 6. Ten spots from UV-Space, UV-Mars, and ground control sample sets that exhibited differential protein expressions were picked and further characterized with mass spectrometry.

FIG. 6.

Comparative analyses of space-returned Bacillus pumilus SAFR-032 spores that were exposed to UV-Space and UV-Mars. These sets of samples were compared with a control that was not exposed to space conditions. Protein spots relevant to functional properties of resistance to space environmental conditions are highlighted. See Results section for explanations. Spot 2: Outer spore coat protein A. Spot 6: glyceraldehyde-3-phosphate dehydrogenase. Spot 16: Superoxide dismutase. Spot 68: transcriptional regulator, AbrB family. Color images available online at www.liebertonline.com/ast

Spots 23, 25, 40, and 68, all of which showed up-regulated proteins for UV-Space, were identified as hypothetical protein BL00422 from B. licheniformis ATCC 14580 (spot 23), alpha-ketoglutarate decarboxylase (spot 25), 30S ribosomal protein S1 (spot 40), and transcriptional regulator, AbrB family of Geobacillus sp. WCH70 (spot 68). The up-regulated proteins for UV-Mars were identified as hypothetical protein BPUM_0788 (spot 19) and serine hydroxymethyltransferase (spot 41). The functionalities of these two proteins are yet to be understood. The spots that showed up-regulated proteins for both UV-Space and UV-Mars were identified as glyceraldehyde-3-phosphate dehydrogenase (spot 6) and superoxide dismutase, respectively (spot 16). Both spots (2, 66) that were up-regulated in ground control spores were identified as the outer spore coat protein A. The outer spore coat protein A (spot 2) was compromised in both the UV-Space–exposed and UV-Mars–exposed spores, whereas the superoxide dismutase (spot 16) was prominent in both the UV-Space and UV-Mars samples when compared with the ground control spores.

4. Discussion

COSPAR planetary protection policies define five planetary protection categories depending on the purpose of the mission and the target body (COSPAR, 2002). Of particular concern for Mars are Category IV (direct contact) and V (sample return) missions. A significant planetary protection challenge for missions to Mars is to achieve surface cleanliness and spacecraft sterility such that it will prevent inadvertent introduction of terrestrial microbes from Earth. The current standard procedures for microbial examination and for cleaning and sterilizing space hardware are largely based on the Viking planetary protection state of knowledge (NASA, 1980). In particular, in the event that some resistant strains of microbes survive the sterilization treatment and are accidentally transported to Mars, researchers must know the microbes' responses to the harsh environment of space and the martian surface in order to avoid false positives in in situ and sample return life detection. This study has provided data, for the first time, on the responses of one of the most resistant bacterial spores (B. pumilus SAFR-032) to the conditions of outer space.

Since the advent of space travel, the possibility of microorganisms surviving in outer space conditions has been examined to determine the upper boundary of the biosphere (Horneck, 1993; Horneck et al., 2010). The quest to determine survivability of microorganisms during interplanetary transfer and establish the limits for survival of life has led to a wide range of real space and simulation experiments on Earth (Horneck, 1993; Horneck et al., 2010). Ground-based setup for space and Mars simulation have helped to define the final layout of space experiments and will clarify phenomena observed in space experiments (Horneck, 1999; Osman et al., 2008b). Such studies were conducted, both singly and in combination, for the following conditions: ultrahigh vacuum with its concomitant extreme desiccation, high and low extremes of temperature, solar UV radiation, cosmic radiation, and microgravity (Nicholson et al., 2000). Most attention has been paid to the effects of simulated extraterrestrial UV radiation because it has been found to be the most deleterious factor encountered in the space environment (Horneck, 1993; Newcombe et al., 2005). When B. subtilis spores were subjected to a Mars simulation chamber (Schuerger et al., 2003, 2006), UV radiation was the most harmful effect on the spores, inactivating them within seconds (lab strains) and minutes (SAFR-032 strain) at the level of a 3 order of magnitude reduction in viability.

Results of the present study indicate that MGR experiments that mimicked UV radiation on the ISS were less lethal to spores than were real space conditions. Additionally, the complex radiation field experienced in outer space cannot be simulated by any ground facility. The sustainability challenges presented by space conditions for microorganisms also include intense galactic and solar radiation, extreme variations in temperature, microgravity, and high vacuum. The effects of these environmental extremes in combination with complex spacecraft surfaces with ample shielding opportunities for microorganisms will be the decisive parameters in spore inactivation (Moores et al., 2007). Hence, in situ exposure to outer space is necessary to provide empirical data on microbial survival mechanisms in extraterrestrial environments.

The majority of published articles on microbial resistance are based on the analyses of laboratory strains (Horneck et al., 1984, 1994, 1995). Hence, researchers currently have a limited understanding and ability to reasonably predict the actual survival and possible adaptation of terrestrial life on real space conditions. The results of this study reinforce that solar UV exposure has the most detrimental impact on viability of highly resistant spores in real space conditions, and that outer space is more detrimental than the martian environment. Prolonged UV radiation (18 months) in real space conditions completely compromised a population of 10⁷ highly UV-resistant B. pumilus spores and left only 19 survivors. The surviving population might be due to any of the following: a resistant subpopulation of spores; partial protection of spores present in multilayers in small groups; or shielding in the hiding places provided by small pits, cracks, and scratches present on the aluminum coupons as seen during this study. It is known that shielding provided by dust particles, thin aluminum foil, or uneven surfaces completely eliminates UV effects (Horneck et al., 2001; Osman et al., 2008a). Likewise, multilayered aggregates of spores on spacecraft materials are known to provide shielding from UV (Schuerger et al., 2003, 2006).

Furthermore, spores that survived real space conditions exhibited enhanced survival (10³ to 10² survivors) when compared with ground-control spores (less than 10 survivors) after exposure to 4000 J/m² UVC; space-surviving spores also showed genetic and proteomic changes. Comparative 2D-DiGE and mass spectrometry protein identification revealed compelling evidence of marked differences in protein profiles between ground-control and space-surviving spores. The alpha-ketoglutarate decarboxylase enzyme (spot 25) is involved in menaquinone biosynthesis, which was further linked to the membrane-associated electron transport system in B. subtilis cells (Palaniappan et al., 1994). However, menaquinone synthesis in spores is not well understood. Protein S1 (spot 40) is the largest ribosomal protein present in the small subunit of the 70S ribosome, and it has been implicated in the selection of the translation start site on the 30S subunit. A pyrimidine-rich region, upstream of the mRNA sequence, interacts with protein S1 and serves as one of the ribosome recognition sites. Protein S1 has been reported to be necessary in some cases for translation initiation and for translation elongation. It is the only ribosomal protein that has a high affinity for mRNA (Sengupta et al., 2001). Research indicates that the AbrB family protein (spot 68) represses various starvation-induced differentiation processes in B. subtilis, among which is competence development (Strauch, 1999). The glyceraldehyde-3-phosphate dehydrogenase, type I (spot 6) that was up-regulated for both UV samples, is reportedly linked to thermostability in spore-forming bacteria (Biesecker and Wonacott 1977; Biesecker et al., 1977). Compared with their ground control counterparts, the second common up-regulated protein in the real space UV-exposed spores was superoxide dismutase (spot 16), which has been shown to catalyze the dismutation of superoxide radicals (McCord and Fridovich, 1969a, 1969b). In addition, sodA and manganese were reported to be essential for resistance to oxidative stress (Inaoka et al., 1999). More molecular analyses are required to detect genetic and proteomic changes in ISS survivor spore adaptation to space conditions.

The data generated are important for assessing the limits and mechanisms of microbial survival, survival strategies for organisms in extraterrestrial environments, microbial contaminants of risk for forward contamination, and in situ life detection. However, the following key questions require in-depth examination: (i) What is the nature and importance of spore DNA damage in SAFR-032 caused by exposure to vacuum, solar radiation, simulated martian surface radiation, and cosmic radiation? (ii) At what rates do these damages accumulate, and how do these accumulation rates correspond to spore inactivation rates? (iii) What specific repair systems are activated during subsequent spore germination? The SAFR-032 spores that were exposed to real space conditions and corresponding ground control specimens are archived in our laboratory. Future experimentations on the space-returned archived samples with the use of emerging molecular techniques will help us to further understand how and why spacecraft-associated microbes might survive as hitchhikers on space vehicles and compromise the science of life-detection and sample return missions.

5. Conclusion

This study has exposed multiresistant bacterial spores to real space and simulated martian conditions for prolonged periods to assess effects on spore viability. The results indicate that UV exposure under space conditions nearly wiped out an entire spore population. Spores managed to survive under dark conditions as well as in the middle and bottom layers of the exposure tray protected from UV. We also observed that spores exposed to space and simulated martian conditions have elevated levels of proteins responsible for resistance traits. A subpopulation of spores may possess enhanced protective machinery and may survive under extreme space conditions. Given our results, we hypothesize that spores sheltered under spacecraft structures, as well as a mutant subpopulation, can survive during space travel. This study provides new insights into the principal limits of life and its adaptation to environmental extremes on Earth or other planets. The research has implications for the evolution and distribution of life.

Footnotes

Acknowledgments

The research described in this publication was carried out in part at the Jet Propulsion Laboratory, California Institute of Technology (Caltech), under a contract with the National Aeronautics and Space Administration. This research was funded by a 2007 NASA Research Announcement Research Opportunities in Space and Earth Sciences (NRA ROSES) grant. The authors acknowledge the technical assistance of students hired through the NASA Undergraduate Student Research Program (A. Chopra), the Caltech Amgen Scholarship (B. Chen), and the JPL Graduate Fellowship Program (P. Schwendner). We are grateful to J. Rummel and C. Conley for useful discussion and to J.A. Spry for encouragement and reviewing the manuscript. The PROTECT team thanks the astronauts who were involved in the exposure and retrieval of EXPOSE-E; the team at ESA's European Space Research and Technology Centre during EXPOSE-E planning, operation, and evaluation; the KT and RedShift teams; and the team at DLR Microgravity User Support Center, who contributed to the EXPOSE-E preparation, engineering verification testing, EST, and MGR. © 2011. All rights reserved.

Abbreviations

COSPAR, the Committee on Space Research; DiGE, difference gel electrophoresis; DLR, German Aerospace Center; EDX, energy-dispersive X-ray; ESA, the European Space Agency; EuTEF, European Technology Exposure Facility; FE-SEM, field-emission environmental scanning electron microscope; ISS, International Space Station; JPL, Jet Propulsion Laboratory; MGR, mission ground reference; NSM, nutrient broth sporulation medium; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PVA, polyvinyl alcohol; TSA, tryptic soy agar.

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Survival of Bacillus pumilus Spores for a Prolonged Period of Time in Real Space Conditions

Abstract

1. Introduction

2. Materials and Methods

2.1. Bacterial strains and purification of spores

2.2. Spacecraft-qualified aluminum coupon preparation

2.3. Spore seeding of the coupons

2.4. Spore recovery from coupons

2.5. Selection of UV254 resistance

2.6. Experiment hardware of the PROTECT experiment on board the ISS

2.7. Mission ground reference conditions

2.8. Microscopy

2.9. Proteomic analyses of “space-return” B. pumilus spores

3. Results

3.1. Morphology of “space-surviving” spores

3.2. Survival assessment of spores exposed to space conditions

3.3. Enhanced UVC resistance of “space-surviving” strains (spores and vegetative cells)

3.4. Proteomic characterization of “space-exposed” B. pumilus spores

4. Discussion

5. Conclusion

Footnotes

Acknowledgments

Abbreviations

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2.5. Selection of UV₂₅₄ resistance