Bacillus pumilus SAFR-032 isolate

Abstract

Spore-forming microbes recovered from spacecraft surfaces and assembly facilities were exposed to simulated martian UV irradiation. The effects of UVA (315-400 nm), UVA+B (280-400 nm), and full spectrum (200-400 nm) on the survival of microorganisms were studied at UV intensities expected to strike the surfaces of Mars. Microbial species isolated from several spacecraft surfaces such as Mars Odyssey, X-2000 [avionics], and the International Space Station and their assembly facilities were identified using 16S rDNA sequencing. Among 43 Bacillus spore lines screened, 19 isolates showed resistance to UVC irradiation after exposure to 1,000 J m−2 of UVC at 254 nm using a low-pressure mercury lamp. Spores of Bacillus species isolated from spacecraft-associated surfaces were more resistant than a standard dosimetric strain, B. subtilis 168. In addition, UVA+B irradiation required a 35-fold greater exposure time to reduce viable spore numbers by 90% reduction than exposure to the full UV spectrum confirming that UVC (200 to 280 nm) is the primary biocidal bandwidth. Among all Bacillus species tested, spores of a B. pumilus strain showed the highest resistance to all three UV bandwidths as well as the total spectrum. The resistance to simulated Mars UV irradiation was strain-specific; B. pumilus SAFR-032 exhibited the highest resistance compared to all other species tested. The isolation of organisms like B. pumilus SAFR-032 and its elevated survival (6 times) compared to that of the standard dosimetric strains should be considered when determining sanitation capabilities of UV irradiation.

Description

INTRODUCTION

The goal of planetary protection as stated in National Aeronautics and Space Administration (NASA) policy is the prevention of forward and backward contamination (36). This policy applies directly to the control of terrestrial organisms contaminating spacecraft intended to land, orbit, flyby or be in the vicinity of extraterrestrial bodies. Viking mission landers were terminally heat-sterilized to decrease the risk of forward contamination to Mars and to assure that terrestrial microorganisms would not contaminate the life detection experiments (36). 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 (7). The non-life detection Mars landing missions such as Mars Exploration Rovers (MER) 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 MER vehicles (8, 37).

The question is whether the forward contamination of Mars will be significantly decreased by the inherent harsh environment at the martian surface. Spores of Bacillus subtilis have been shown to survive up to 6 years under low-Earth orbit conditions (12, 14). However, only shielding from UV radiation enabled B. subtilis endospores to survive the conditions long-term (12, 15). The solar flux at the martian surface is considerably less than that experienced in interplanetary space (12, 39) and there is the potential that atmospheric conditions could further attenuate UV irradiation (6, 9).

Ultraviolet irradiation has been used to decontaminate or sterilize surfaces, air, and water (5, 16, 34, 46). Dormant spores of various Bacillus and Clostridium species are much more resistant than their vegetative cell counterparts to a variety of treatments including heat, UV radiation, and oxidizing agents such as H₂O₂ (41). Previous work has demonstrated that the binding of α/β-type small acid soluble proteins to spore DNA is the predominant if not the sole determinant of spore UV resistance (40). Further studies using B. subtilis spores implicated several genes, dacB, spl, and ssp, as essential in UV irradiation resistance (31). In addition, the spore coat has been shown to offer intrinsic protection from UVA and B (33). However, few reports are available that test UV reistance on a diverse range of environmental strains of Bacillus species (24). Invariably all microbial lethality assessments and sterilizer validations have been carried out using laboratory strains of B. subtilis and/or Geobacillus stearothermophilus (27). Microbes inhabiting these nutrient poor and dry spacecraft assembly facilities and their resistance traits are important to aerospace, medical and pharmaceutical industries. These resistances could enable the organisms to escape sterilization protocols and possibly survive to contaminate planets or other critical industrial implements.

In this study, we screened endospores of isolated spore forming bacteria from a growing collection of spacecraft and associated environmental isolates for their resistance to UV₂₅₄ irradiation. In order to examine the biocidal effects of direct UV irradiation predicted for Mars, UV₂₅₄ resistant spores were exposed to simulated martian UV irradiation. The effects of UVA, UVA+B, UVC, and the total UV irradiation on the survival of microorganisms, at intensities expected to strike the surface of equatorial Mars.

Materials and Methods

Isolation and identification of microbes from spacecraft surfaces and associated assembly facilities. Species of Bacillus were previously isolated from surfaces of Mars Odyssey and X-2000 (avionics) spacecraft and their associated assembly facilities at the Jet Propulsion Laboratory (JPL), Kennedy Space Center (KSC) and BAE Systems (Manassas, Va.). Bacillus species were also isolated from surface samples taken in the International Space Station. Surface areas of approximately 25 cm² were sampled using water-moistened, sterile polyester swabs (Catalogue no. TX761; Texwipe). Selected floor surfaces measuring approximately 3,600 cm² were sampled using sterile wipes (Cat. no. TX 1009; Texwipe). Each swab or wipe sample was placed into sterile polypropylene bottles containing 10 or 100 mL sterile water, respectively, and transferred to the laboratory for analysis. Swabs exposed to the assembly facility atmosphere but not used in active sample collection served as controls. The spore-forming bacterial populations were selected by sonicating the samples for 2 min and heat-shocking at 80° C. for 15 min. Appropriate aliquots of samples were placed into petri dishes, in duplicate, using the pour plate technique with trypticase soy agar (TSA; Becton Dickinson and Co.) as a growth medium. Following a 2-day incubation at 32° C. (1, 2) isolates were selected, purified and stored at −80° C. for further processing and analysis. All other strains were purchased from the American Type Culture Collection (ATCC) and listed in Table 1.

Identification of purified strains was determined based on 16S rDNA sequencing. Bacterial small subunit rRNA genes were PCR-amplified with eubacterially biased primers B27F and B1492R (22). PCR conditions were followed as described elsewhere (35). The PCR-amplified 16S rDNA fragments were purified using Qiaquick columns (Qiagen) and were fully, bi-directionally sequenced. The phylogenetic relationships of organisms covered in this study were determined by comparison of individual 16S rDNA sequences to sequences in the public database described at http://www.ncbi.nlm.nih.gov. Evolutionary trees were constructed via phylogenetic analysis using parsimony software described at http://paup.csit.fsu.edu. Sources of the tested strains along with their 16S rDNA sequences and GenBank accession numbers are given in Table 1.

Sporulation. A nutrient broth sporulation medium (NSM) was used to initially sporulate test bacteria (26, 38). A single purified colony was inoculated into NSM liquid medium, incubated at 32° C. and the cultures examined via wet mounts and light microscopy each day to determine the level of sporulation. Once the number of free spores in each culture was greater than 99% of the total number of cells present, typically 2-3 days, cultures were harvested via centrifugation. Spore purification was performed by treating the spores with lysozyme and washing with salts and detergent, as described by Nicholson and Setlow, 1990 (26). Purified spores were resuspended in sterile deionized water, heat-shocked at 80° C. for 15 min, and stored at 4° C. in glass tubes until used.

Selection of UV₂₅₄ resistant spores. Purified spores of 44 strains (Table 1) were diluted in phosphate-buffered saline (PBS, pH 7.2) to a density of 10⁶ per mL. Initial spore density was estimated by the dilution plating method before each exposure. A low pressure handheld mercury arc-UV lamp (UVP, Inc.; model UVG-11) was placed over the sample and UV flux at the surface of the spore suspension was measured using a UVX digital radiometer (UVP, Inc.). The exposure time to produce 1,000 J m⁻² of energy at the sample surface was determined as 167 sec at 600 μW cm⁻². The spore suspension was placed in an uncovered 100 mm glass petri dish containing a magnetic stir bar and exposed to UV₂₅₄ irradiation under sterile conditions. In a qualitative screen, strains surviving 1,000 J m⁻² irradiation were selected for quantitative lethal dose curve analysis. Sample volumes of 100 μL were removed after specific periods of time, serially diluted and plated on TSA. Spores that maintained LD₉₀, a dose at which 90% of the spores inactivated, values >200 J m⁻² were chosen as resistant for further experiments.

Mars atmospheric radiation model. FIG. 1 compares the output of a Mars atmospheric irradiation model with the spectral irradiances used in the lab experiments. The UV Mars atmosphere model used in this study was developed using Mars Pathfinder data for visible light to near infrared and atmospheric transmittance models developed at the University of Arizona Lunar and Planetary Lab (42). Expansion of the model into the UV bandwidth was achieved by extrapolation and comparing the results at specific wavelengths to observational data for Mars dust-like materials (unpublished data). Four conditions were modeled to explore the range of UV irradiance from a likely lander site (FIG. 1). Two levels of dust were modeled, optical density of 0.24 and 0.74, as measured at 671 nm. These correspond to the range of dust levels experienced by the Opportunity rover (4). The high sun conditions were typical of a lander with the sun directly overhead, a solar zenith angle of 2.06 degrees. The low sun condition simulated a position 45 degrees south during the martian winter, with the same two optical densities of dust. Here the solar zenith angle was 70.2 degrees, which significantly reduced the UV irradiances due to dust absorption. The spectral shape is very similar between the four model spectra since the shape of the spectrum is affected by dust absorption/scattering. Ozone absorption or ice scattering was not modeled as these are not the most common conditions.

Resistance of spores in water to simulated Mars UV irradiation. Spores resistant to UV₂₅₄ irradiation were exposed to UVA (315-400 nm), UVA+B (280-400 nm), or total UV (200-400 nm) at the Mars simulated solar constant of 590 W m⁻² total irradiance (190 nm to 3 μm) (3) using a X-25 solar simulator (Spectrolab Inc.) equipped with a xenon arc lamp located at the Environmental Test Laboratory, JPL. The intensity of UV irradiation was approximately 10% of the full spectrum (59.0 W m⁻²). The total irradiance level in these simulations matched the actual Mars solar constant of 590 W m⁻² (3). The irradiation intensity was constantly adjusted by fine-tuning the lamp power to achieve ±5% variability as indicated by an Optronics Laboratories OL754 spectroradiometer. The various bandwidths were generated by placing Corning glass (UVA) or plastic petri dish lids (UVA+B, Fisher Scientific) as filters in front of the sample cuvettes. The lethal dosages (LD) values were calculated for each strain to estimate LD₅₀, LD₉₀, and LD₁₀₀ (or D value) UV dosages. Lethal Dose₁₀₀ values were defined as the UV dosage in which no cultivable spores were recovered using the procedures described herein.

Spores of nine strains exhibiting the highest LD₉₀ values were diluted with sterile deionized water to approximately 1×10⁶ spores mL⁻¹. Then, 2-mL aliquots of the suspensions were placed in 3-mL Suprasil quartz cuvettes (10 mm path length) equipped with a 3×3 mm micro-stirbar (Fisher Scientific). When two strains were mixed for exposure, 5×10⁵ spores mL₋₁ of each species were mixed to a final density of 10⁶ spores mL⁻¹. UV exposure times ranged from periods of 30 sec to 30 min. At various intervals, 100 μL samples were removed, diluted serially tenfold in sterile PBS, and plated onto TSA. All TSA plates were incubated at 32° C. for 24-48 h and colony-forming units were enumerated. The most resistant spores were selected for further experimentation. Quartz cuvettes were cleaned after each exposure experiment by rinsing 3 times with 70% ethanol followed by 3 rinses of 95% ethanol. The rinsed and dried cuvettes were placed in appropriate gas-permeable envelopes (Tyvek® pouches, Advanced Sterilization Products) and sterilized after exposure to 1 to 4 cycles of hydrogen peroxide injections in a Sterrad 100S vapor hydrogen peroxide sterilizer (Advanced Sterilization Products).

In addition to the X-25 system spores of two strains, B. subtilis 168 and B. pumilus SAFR-032, were exposed to simulated martian irradiation using a xenon arc lamp (model 6262, Oriel Instruments) located at KSC under spectral conditions reported previously (12, 39). Various bandwidths were generated as above. In all UV assays that examined a mixture of B. subtilis 168 and B. pumilus SAFR-032 enumeration was possible due to the distinctly different colony morphologies of the two organisms while growing on agar medium.

Results

Identification of UV irradiation resistant microbes. Initially, spores of 43 strains isolated from several spacecraft surfaces and associated assembly facilities (Table 1), and survived a heat shock protocol were screened for UVC₂₅₄ resistance using a Hg lamp. Nineteen strains exhibited growth after receiving a dose of 1,000 J m⁻². Phylogenetic analyses were performed on all the strains tested; they were unambiguously determined to be low G+C Gram-positive Fermicutes based on 16S rDNA sequence analysis. The 16S rDNA sequences of all isolates were compared and bootstrapping (500 replicates) analysis was performed to avoid sampling artifacts. The resulting analyses indicated that the tested strains shared a close phylogenic relationship with Bacillus species. Neighbor-joining, parsimony, and maximum likelihood analyses were performed on this subset of bacteria using several subdomains of the 16S rDNA. A maximum-likelihood phylogenetic tree based on 16S rDNA sequences of several Bacillus species is shown in FIG. 2. The branching order of this tree showed 2 distinct clusters in which the top clade consisted of Bacillus rRNA group 1 and the other stock was formed by species of rRNA group 2 as well as the exosporium-like producing Bacillus species of rRNA group 1 (32).

The similarities in 16S rDNA nucleotide sequence between the strains tested in this study and closely related Bacillus species, recognized by GenBank “BLAST” searches, were between 91.6 and 99.8%. A sequence variation of <3% was observed between B. subtilis, B. mojavensis, B. atrophaeus, B. licheniformis, and B. pumilus (FIG. 2). The strains of the B. subtilis and B. pumilus sequenced in this study were tightly bound phylogenetically, as all shared 16S rDNA sequence similarity >97.5%. Additionally, a high 16S rDNA sequence variation (−8%) was observed between members of rRNA 1 and rRNA 2 groups; however, a high degree of dissimilarity within a well-described genus is not uncommon. Based on 16S rRNA analysis, five of the tested environmental strains were identified as members of the rRNA group 1, B. pumilus FO-33, FO-36b, SAFN-036, SAFR-032, and B. subtilis 42HS-1 and two, B. odysseyi and B. psychrodurans VSE 1-06, were identified as belonging to rRNA group 2 (Table 1).

Martian UV irradiation simulation. The JPL and KSC lab spectra were very similar in shape and level to the model, with the KSC spectrum more representative (FIG. 1). The JPL spectrum was deficient in 250 nm energy and exhibited a significant dip at 330 nm. This resulted in lower energies in the UVC and UVA bands, with very similar levels for the UVB band. The 330-nm dip seen in the JPL spectrum was most likely due to lens coatings within the solar simulator, but was not deemed critical because most of the absorption band was in the UVA region of the solar simulation. Our results (shown below) indicated that UVA had minimal effect on the survival of bacterial spores. The JPL and KSC simulations of the Mars solar environments were calibrated to fall within a range of UV irradiation encountered under clear-sky conditions, optical depth <1, for the Mars rover Opportunity at high and low sun angles.

Survival of endospores in aqueous solution under simulated Martian UV irradiation conditions (JPL simulation). Of the 19 strains that exhibited UVC resistance, seven spacecraft associated isolates were chosen for further study due to their elevated LD₉₀ values in response to UV irradiation (data not shown) or other traits. The bacterial strains and determining factor selected for exposure to simulated Mars UV irradiation were as follows: B. odysseyi, for its morphological novelty (21); B. psychrodurans VSE1-06, for its low temperature tolerance; 4 strains of B. pumilus, for predominant occurrence; and B. subtilis 42HS-1, a close relative of the well-studied reference Bacillus subtilis 168 (5, 11). In addition to the above environmental strains, B. subtilis 168 was selected as a control since this strain has been used in numerous other resistance studies that have been previously published (10, 11, 13, 14, 28, 39). Similarly, B. megaterium ATCC 14581 was also chosen for further study due to its high UV resistance when compared to other reference strains used in the current work. All of the B. pumilus environmental isolates were obtained from the JPL spacecraft assembly facility class 100K cleanrooms except B. pumilus 015342-2 ISS which was isolated from surfaces of the ISS. Both B. subtilis 42hs- 1 and B. odysseyi were cultured from the surface of the Mars Odyssey and B. psychrodurans VSE 1-06 was recovered from air samples collected within the Mars Exploration Rovers assembly facility, Payload Hazardous Servicing Facility (PHSF), KSC (Table 1).

The results of exposing Bacillus spores in aqueous solution to UVA, UVA+B, and total UV are shown in Table 2. When compared to the full UV spectrum, a 2- to 25-fold increase in exposure time of UVA or A+B was required to reduce 50% of the viable spore counts. Likewise, 90% reductions in viable spore numbers required 35-140 times greater exposure time to UVA+B or A, respectively, than to the full UV spectrum. As shown in Table 2, none of the Bacillus species tested were completely eradicated even after 30 min exposure to UVA+B irradiation or UVA (Table 2). The LD₅₀, LD₉₀, and LD₁₀₀ of the nine bacterial spores tested under various UV spectra of the Mars solar simulation showed that UVA+B irradiation was significantly less lethal than full spectrum UV irradiation, therefore as expected the 200-280 nm range is more damaging than longer wavelengths. Although all spores tested exhibited sensitivity to UVA+B, most damage by UVA+B might be attributed to UVB. This was further confirmed by the observation that all the spores tested, except B. pumilus FO-033, were resistant to UVA with growth observed even after 30 min exposure (Table 1).

Based on the Bacillus species tested, resistance to UV A, A+B or full spectrum was found to be strain-specific. However, for Mars full UV spectra, three out of four B. pumilus strains tested in this study exhibited LD₅₀ values of 40 to 80 sec and LD₉₀ values of 100 to 270 sec. Except for B. pumilus SAFR-032 and B. megaterium, spores of the other Bacillus species tested showed LD₅₀, LD₉₀ and LD₁₀₀ values as <24 sec, <60 sec, and <120 sec, respectively, for Mars full UV spectra. Furthermore, B. pumilus SAFR-032 spores that showed resistance to 2,000 J m⁻² UV₂₅₄ (24) were not completely killed after 30 min when exposed to full UV spectrum under Mars simulated intensities. In addition, SAFR-032 spores showed a greater resistance, LD₅₀ of 84 sec and LD₉₀ of 270 sec, than the other strains to full UV exposure at the Mars simulated solar UV irradiation conditions. The reference strain B. subtilis 168, exhibited LD₅₀, LD₉₀ and LD₁₀₀ values under full UV spectra of 24 sec, 42 sec, and 72 sec, respectively.

Survival rate plots of B. pumilus SAFR-032, B. megaterium, and B. subtilis 168 at various time intervals under Mars full UV irradiation are shown in FIG. 3. Bacillus pumilus SAFR-032 spores exhibited classical inactivation kinetics with a characteristic “shoulder” extending to 2 min followed by a strict exponential inactivation plot thereafter. However, B. subtilis 168 and B. megaterium spores experienced a sharp decline in viability immediately after 30 sec of UV exposure. After a 10 min UV exposure, the cultivability of B. megaterium spores was completely lost while a portion of B. pumilus SAFR-032 spores survived.

Effects of various UV spectra under Mars solar UV irradiation conditions on the survival of B. pumilus SAFR-032 spores are shown in FIG. 4. Neither UVA and UVA+B radiation was effective in inactivating SAFR-032 spores, even after 30 min exposure, whereas the full simulated spectrum reduced SAFR-032 spore viability by >3 orders magnitude after 30 min irradiation. When initial concentrations of spores were reduced from 10⁶ mL⁻¹ to 10⁵ mL⁻¹ or 10⁴ mL⁻¹ before UV exposure, the spores of B. pumilus SAFR-032 were completely killed in 8 and 2 min, respectively (data not shown).

The influence of viable and heat-killed SAFR-032 spores on the UV sensitive B. subtilis 168 spores was tested (FIG. 5). Equal proportions of viable spores of B. pumilus SAFR-032 and B. subtilis 168 were mixed to a final density of approximately 10⁶ spores mL⁻¹ (5×10⁵ spores mL⁻¹ of each strain) and exposed to full simulated martian UV irradiation. The time taken to eradicate all B. subtilis 168 when mixed with equal parts of B. pumilus SAFR-032 spores was ˜17 min as opposed to 1.2 or 5 min when B. subtilis 168 spores at concentrations of 5×10⁵ or 1×10⁶ spores mL⁻¹, respectively, were exposed alone (FIG. 3; Table 2). Heat-killed B. pumilus SAFR-032 spores offered no protection to B. subtilis 168 (data not shown).

Vegetative cells of B. pumilus SAFR-032, B. subtilis 168 and Acinetobacter radioresistens 50 v1, isolated from Mars Odyssey, were exposed to UV₂₅₄ at a rate of 1 J m⁻² sec⁻¹ and recovered under lighted conditions (FIG. 6). The results indicate that all three strains exhibited similar inactivation rates up to 50 J m⁻² total dose after which cells of B. pumilus SAFR-032 and A. radioresistens survived a 200 J m⁻² dose (only 3-logs reduction) while B. subtilis 168 cells were rendered unrecoverable after 50 J m⁻².

Survival of endospores in aqueous solution under simulated Martian UV irradiation conditions (KSC simulation). Since several publications have shown variations in martian UV simulations (6, 18, 39) we UV-irradiated spores of B. pumilus SAFR-032 and B. pumilus 168 using an alternate martian UV simulation system developed at KSC (34). Spores of these organisms were exposed to martian UV, UVA, UVA+B and full spectrum irradiation conditions. Aliquots of UV exposed spores were plated onto TSA after UV exposures of 0, 1, 2, 5, 10, and 20 min and surviving spore-forming cells were counted after appropriate cultural conditions. FIGS. 7(A, B, and C) shows the inactivation curves of the spores of SAFR-032 and 168 at full Mars UV, UVA+B, and UVA, respectively. The LD₉₀ of B. subtilis 168 spores was <30 sec when exposed to full martian UV irradiation and SAFR-032 spores showed at least 3 times higher resistance than 168 spores (FIG. 7A). Under the total UV spectrum the cultivability of B. subtilis 168 spores was lost after approximately 90 sec while B. pumilus SAFR-032 showed a 2-3 times greater resistance, remaining cultivable after 5 min (FIG. 7A). Under UVA+B exposure, the survival of B. pumilus SAFR-032 dramatically increased with 90% of the spores maintaining cultivability while B. subtilis 168 was almost rendered non-cultivable after 20 min exposure (FIG. 7B). Similar results were observed under UVA irradiance; however, B. subtilis 168 maintained cultivability after 20 min. (FIG. 7C). It should be noted that the capability did not exist to tune the KSC xenon arc lamp in real time therefore when filters were placed in front of the light path a decrease in UV fluence of 20-40% occurred (47). This decrease in fluence partially explains the enhanced resistance to these two wavelengths during the experiment. However, even at the decreased fluences B. pumilus SAFR-032 maintained greater resistance than B. subtilis 168.

Discussion

During several surveys on the microbial diversity of spacecraft assembly facilities over a period of 3 years, 125 aerobic microbial strains have been isolated (17, 20, 21, 43, 44) and their phylogenetic affiliations characterized (19, 45). Eighty five percent of these strains were identified as Gram-positive bacteria. About 65% of the total strains isolated survived heat-shock protocols (2). Members of the Bacillus genus were the most predominant microbes among the heat-shock survivors (>91 %). In total 15 different Bacillus species were identified. B. licheniformis was the most prevalent (25%) while B. pumilus (16%) was the second-most prevalent species isolated from spacecraft assembly facilities.

Previous UV resistance studies have utilized model dosimetric strains and indicated the limit to survival for organisms as approximately 200 J m⁻² UV₂₅₄ (30). A recent study examined the survival of a laboratory strain, B. subtilis HA 101, on spacecraft qualified materials under simulated martian UV irradiance (39). The results suggested that ˜6-logs of the spores exposed on spacecraft surfaces under the simulated UV were inactivated within a few tens of minutes under Mars equatorial and clear-sky, optical depth of 0.5, conditions. Other researchers have examined a B. pumilus strain, isolated from a spacecraft assembly facility, and reported it maintained one of the highest UV₂₅₄ resistances reported for spores to date (24). Since most of the published UV resistance information has involved the use of laboratory strains, predicting the actual survival and possible adaptation of terrestrial life on Mars is limited due to the lack of robust empirical data. This 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 standards for UV disinfection of drinking water are set at 400 J m⁻² UV 254. B. pumilus SAFR-032 would require doses of 2000-2500 J m⁻², an order of magnitude greater than the standard, for complete sanitation (25).

The current study is the first to report the survival capabilities of other commonly occurring bacterial spore-forming microorganisms, recovered from spacecraft surfaces, to simulated martian UV exposures. The data presented here indicates spores of B. pumilus SAFR-032 are far more resistant to simulated martian UV irradiation conditions than standard dosimetric strains. 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 will be 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 protected the more UV sensitive B. subtilis 168 spores. Specifically, colonies of B. subtilis 168 were not observed on plates following treatments of ˜2 min or longer with martian UV irradiation. However, when mixed with B. pumilus SAFR-032, spores of B. subtilis 168 survived 5 or 10 min of martian UV irradiation (FIG. 5). Autoclaved B. pumilus SAFR-032 spores did not exact any protective properties to B. subtilis 168 spores and the inactivation curve for spores of B. subtilis 168 was very similar to those generated by unmixed 168 spores at densities 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 the 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 simulation used by Schuerger et al. (39), as seen in FIG. 1. 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 their chemical and physical composition. From the UV spectral irradiance of both JPL and KSC, it is evident that the KSC UV simulation exhibited a higher UV flux below 260 nm and was more lethal to both organisms tested. Ultraviolet irradiation below 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 (15, 23, 29). Since the KSC simulator was more rich in UVC than that of JPL, the difference in inactivation rates reiterate that UV irradiation in the 200-280 nm range is the 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 most sun-exposed microorganisms would be rapidly inactivated at equatorial latitudes. However, existence of organisms like SAFR-032, the survival of which was significantly greater than that of the standard lab strain, B. subtilis 168, should be considered when examining 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 non-biological spacecraft residues (e.g., lubricants) on the survival of terrestrial microorganisms under martian UV fluence rates, and (iii) to determine if low levels of diffuse UV irradiation will permit the adaptation of terrestrial microorganisms to differing martian conditions. The research described herein has demonstrated that the Mars UV environment is likely to be very detrimental to the survival of microbial species from Earth, but until these other questions can be properly addressed we must remain vigilant in processing spacecraft for Mars to reduce the possibility of forward contamination of landing sites.

ACKNOWLEDGEMENTS

Part of the research described in this publication was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration. This research was funded by a Director's Research Discretionary Fund (100656-001057) awarded to Dr. Venkateswaran. We are grateful to members of the Biotechnology and Planetary Protection group for technical assistance and collecting the spacecraft-associated microbial strains. The authors thank P. Martin for assistance with Mars solar UV simulations at JPL, M. Anderson for Mars solar UV calibrations at JPL, M. T. La Duc for 16S rDNA sequence analysis, and W. Nicholson and G. Homeck for critically reading the manuscript. We also appreciate the help rendered by J. Moores for running the Mars UV simulation models and P. Smith for the development of the Mars UV models. We are thankful to K. Buxbaum, and G-S. Chen for valuable advice and encouragement.

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FIGURE LEGENDS

1. Spectral irradiance plots of the JPL solar simulator (dashed line); KSC simulator (solid line); the Opportunity sky conditions of OD=0.24, high sun (diamonds); OD=0.74, high sun (squares); and predicted sky conditions for latitude 45S, OD=0.24, low sun (circles) and latitude 45S, OD=0.74, low sun (triangles).

2. Phylogenetic tree of the type strains of Bacillus spp. strains used in this study based on maximum likelihood parsimony analysis of the 16S rDNA nucleotide sequences. The numbers after the names of the bacteria are the GenBank nucleotide accession numbers. Numbers above the lines are the percent bootstrap values of 500 replications of that branch of the tree.

3. Effect of Mars UV irradiation (JPL simulator) on the hydrated spores of select Bacillus species: B. pumilus SAFR-032 (closed square), B. megaterium ATCC 14581 (closed circle), andB. subtilis 168 (open triangle). The error bars represent the standard deviations of 3 replicate samples.

4. Influence of various Mars UV spectra on the survival of B. pumilus SAFR-032 spores in water (JPL simulator); UVA (closed circle), UVA+B (open triangle), and full UV (closed squares). Error bars represent the standard deviations of 3 replicate samples.

5. Effect of B. pumilus SAFR-032 on spores susceptible to UV-induced inactivation (JPL simulator); B. subtilis 168 in mixture (closed square), B. subtilis 168 alone (open triangle), and B. pumilus SAFR-032 alone (closed circle). In the mixed samples equal amounts of spores from both species were mixed to yield a total spore density of 106 ml⁻¹. Error bars represent the standard deviations of 3 replicate samples.

6. Effect of UV₂₅₄ irradiation on vegetative cells: B. pumilus SAFR-032 (closed square), A. radioresistens (closed circle), and B. subtilis 168 (closed triangle). The error bars represent the standard deviations of 3 replicate samples.

7. Effect of Mars UV irradiation, projected from the KSC simulator, on the spores of select B. pumilus SAFR-032 (closed square) and of B. subtilis 168 (closed square) in water. Three conditions of full spectrum UV, UVA+B, and UVA are represented in panels A, B, and C respectively. The error bars represent the standard deviations of 3 replicate samples.

TABLE 1
Various characteristics of microbes associated with several spacecraft and associated environments tested in this study
					Qualitative
				GenBank accesion	Resistance to
Species	Strain1	Source	Location	no.	UV254	Reference
B. atrophaeus	ATCC 9372	Raven Lab	NASA standard strain	X60607	−−6	32
			(formerly known as B.
			globigii)
B. benzoevorans	ATCC 49005T	ATCC	Soil in France	D78311	−−	32
	SAFN-024	Clean room floor	JPL-SAF	AY167808	++	This study
B. cereus	ATCC 14579T	Air	ATCC	AF290547	++	32
	FO-11	Clean room air particulates	JPL-SAF2	AF234842	−−	17
B. firmus	ATCC 14575T	ATCC	N/A3	X60616	++	32
	10V2-2	Clean room floor	KSC-PHSF4	AF526919	++	19
B. flexus	ES-16	Clean room table	Manassas, VA	N/A	−−	This study
B. fusiformis	ATCC 7055T	ATCC	N/A	L14013	++	32
B. gibsonii	ATCC 700164T	ATCC	N/A	X76446	−+	32
B. licheniformis	KL-196	Clean room floor	JPL-Vibration Testing	AF387515	++	29
			Facility
B. megaterium	ATCC 14581T	ATCC	Milk	X60629	++	32
	KL-197	Entrance floor	JPL-Highbay	AY030338	−−	29
B. mojavensis	ATCC 51516T	ATCC	Soil, Mojave Desert,	AB021191	−−	32
			Rosamond, CA
	KL-154	Entrance floor	JPL-MECA Facility5	AY030334	−−	29
B. mycoides	ATCC 6462T	ATCC	Soil	X55061	++	32
	FO-080	Clean room air particulates	JPL-SAF	AF234860	−−	17
B. nealsonii	BAA-519T	Clean room air particulates	JPL-SAF	AF234863	−−	30
B. neidei	NRRL BD-101T	USDA	Soil	AF169508	++	19
B. niacini	51-8C	Clean room floor	KSC-PHSF	AF526905	++	17
B. odysseyi	PTA-4993T	Mars Odysseyi spacecraft	KSC-PHSF	AF526913	++	19
B. psychrodurans	VSE1-06	Clean room air particulates	KSC-PHSF	AJ277983	++	This study
B. pumulis	ATCC 7061T	ATCC	N/A	AB020208	−+	32
	ATCC 27142	ATCC	N/A	AY876287	−−	32
	51-3C	Clean room floor	KSC-PHSF	AF526907	−−	19
	82-2C	Clean room floor	KSC-PHSF	AF526902	++	19
	84-1C	Clean room floor	KSC-PHSF	AF526898	−−	19
	84-3C	Clean room floor	KSC-PHSF	AF526896	−+	19
	84-4C	Clean room floor	KSC-PHSF	AF526895	−−	19
	FO-033	Clean room air particulates	JPL-SAF	AF234851	−−	17
	FO-36b	Clean room air particulates	JPL-SAF	AF234854	++	17
	KL-052	Clean room cabinet top	JPL-SAF	AY030327	++	17
	SAFN-027	Clean room	JPL-SAF	AY167884	++	17
	SAFN-029	Clean room	JPL-SAF	AY167883	−+	17
	SAFN-036	Clean room	JPL-SAF	AY167881	++	17
	SAFN-037	Clean room	JPL-SAF	AY177880	−−	17
	SAFR-032	Clean room	JPL-SAF	AY167879	++	17
	015342-2 ISS	Hardware	International Space	AY876228	−−	This study
			Station
B. sphaericus	ATCC 14577T	ATCC	N/A	L14010	++	32
B. subtilis	ATCC 6051T	ATCC	N/A	X60646	++	32
	168	Wayne Nicholson,Univ. of	Full genome sequence	NC000964	++	32
		Arizona
	42HS-1	Clean room floor	KSC-PHSF	AF526912	++	19
B. thuringiensis	ATCC 10792T	ATCC	Mediterranean flour moth,	X55062	−−	32
			Ephestia kuhniella
	SAFN-003	Entrance floor	JPL-SAF	AY167823	++	This study
1Superscript “T” denotes type strain
2JPL-SAF, Jet Propulsion Laboratory, spacecraft assembly facility (Building 179)
3Not available
4KSC-PHSF, Kennedy Space Center, payload hazardous service facility
5JPL-MECA Facility, Jet Propulsion Laboratory, Mars Environmental Chamber Assembly Facility
6Growth was categorized in liquid cultures by OD600 readings (left +/−) and on agar medium by colony counts (right +/−). A (+) symbol indicates: OD600 reading > 0.4 or >30 colonies after 24 hr.

TABLE 2
Time (minutes) taken to reduce various Bacillus spores that are exposed
to various UV radiation conditions at the Mars equatorial solar constant
	Full UV (200 to 400 nm)	UVA + B (280 to 400 nm)	UVA (315 to 400 nm)
Bacillus species	LD50	LD90	LD100	LD50	LD90	LD100	LD50	LD90	LD100
B. pumulis	SAFR-032	1.5	4.5	>30*	10.0	>30	>30	8.0	>30	>30
B. megaterium	ATCC 14581	0.6	2.2	12.0	8.0	23.8	>30	7.9	>30	>30
B. pumilus	FO-036b	0.1	0.2	5.0	2.5	19.0	>30	5.0	28.0	>30
B. pumulis	SAFN-036	0.2	0.6	3.5	1.5	17.0	>30	5.1	>30	>30
B. odysseyi	34HS-1	0.3	0.8	3.5	1.8	12.8	>30	1.5	27.0	>30
B. pumulis	FO-033	0.6	1.6	10.0	1.2	2.0	>30	1.5	1.0	>20
B. subtilis	168	0.3	0.9	5.0	3.4	15.0	>30	1.5	12.6	>30
B. subtilis	42HS-1	0.4	1.4	9.5	4.0	16.2	>30	1.8	16.0	>30
B. psychrodurans	VSE1-06	0.3	0.8	5.0	6.0	23.8	>30	2.8	>30	>30
*Organisms that survived longer than the maximum exposure time are listed with a > sign.

Claims

1. An isolated biologically pure culture of Bacillus pumilus SAFR-032 under GenBank accession number AY167879.