MATERIALS AND METHODS
Isolation and identification of microbes from spacecraft surfaces and associated assembly facilities.
Species of Bacillus were previously isolated from surfaces of the 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 (catalog no. TX761; Texwipe). Selected floor surfaces with areas of approximately 3,600 cm2 were sampled using sterile wipes (catalog no. TX1009; Texwipe). The swab and wipe samples were placed into sterile polypropylene bottles containing 10 and 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 them 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 the growth medium. Following 2 days of 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 and are listed in Table 1.
The identities of purified strains were determined by using 16S rRNA gene sequencing. Bacterial small-subunit rRNA genes were PCR amplified with eubacterially biased primers B27F and B1492R (26). PCR conditions described elsewhere were used (41). The PCR-amplified 16S rRNA gene fragments were purified using Qiaquick columns (QIAGEN) and were fully, bidirectionally sequenced. The phylogenetic relationships of organisms covered in this study were determined by comparison of individual 16S rRNA gene sequences to sequences in the public database described at http://www.ncbi.nlm.nih.gov. Evolutionary trees were constructed by phylogenetic analysis using parsimony software described at http://paup.csit.fsu.edu. The sources of the strains tested and their 16S rRNA gene sequences and GenBank accession numbers are shown in Table 1.
A nutrient broth sporulation medium was used for the initial sporulation of test bacteria (30, 45). A single purified colony was inoculated into liquid nutrient broth sporulation medium and incubated at 32°C, and the cultures were examined by using 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 to 3 days, cultures were harvested by centrifugation. Spore purification was performed by treating the spores with lysozyme and washing them with salts and detergent, as described by Nicholson and Setlow (30). 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 they were used.
Selection of UV254-resistant spores.
Purified spores of 44 strains (Table 1) were diluted in phosphate-buffered saline (pH 7.2) to obtain a density of 106 spores per ml. The initial spore density was estimated by the dilution plating method before each exposure. A low-pressure handheld mercury arc UV lamp (model UVG-11; UVP, Inc.) was placed over a sample, and the UV flux at the surface of the spore suspension was measured using a UVX digital radiometer (UVP, Inc.). The exposure time required to produce 1,000 J m–2 of energy at the sample surface was determined to be 167 s at 600 µW cm–2. The spore suspension was placed in an uncovered 100-mm glass petri dish containing a magnetic stir bar and was exposed to UV254 irradiation under sterile conditions. In a qualitative screening analysis, strains surviving 1,000 J m–2 irradiation were selected for quantitative lethal dose curve analysis. Samples (100 µl) were removed after specific periods of time, serially diluted, and plated on TSA. The dose at which 90% of the spores were inactivated (LD90) was >200 J m–2 were considered resistant and were used for further experiments.
Mars atmospheric radiation model.
Resistance of spores in water to simulated Mars UV irradiation.
Figure 1 compares the output of a Mars atmospheric irradiation model with the spectral irradiances used in the lab experiments. The Mars atmosphere UV model used in this study was developed using Mars Pathfinder data for visible light to near-infrared light and atmospheric transmittance models developed at the University of Arizona Lunar and Planetary Lab (51). Expansion of the model into the UV bandwidth was accomplished by extrapolation and by 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 for a likely lander site (Fig. 1). Two levels of dust were modeled (optical densities at 671 nm of 0.24 and 0.74), and these levels correspond to the range of dust levels experienced by the Opportunity rover (4). The high-sun conditions were typical for a lander with the sun directly overhead, a solar zenith angle of 2.06°. The low-sun conditions simulated a position 45° south during the Martian winter, with the same two optical densities of dust. Here the solar zenith angle was 70.2°, which significantly reduced the UV irradiance due to dust absorption. The spectral shapes are very similar for the four model spectra since the shape of a spectrum is affected by dust absorption and scattering. Ozone absorption or ice scattering was not modeled as these conditions are not the most common conditions.
Spores resistant to UV254
irradiation were exposed to UVA (315 to 400 nm), UVA+B (280 to 400 nm), or total UV (200 to 400 nm) at a simulated Mars constant level of total solar irradiance of 590 W m–2
(190 nm to 3 µm) (3
) using an 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 intensity of the full spectrum (59.0 W m–2
). The total level of irradiance in these simulations matched the actual Mars constant level of solar irradiance, 590 W m–2
). The intensity of the irradiation 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 using Corning glass (UVA) or plastic petri dish lids (UVA+B; Fisher Scientific) as filters in front of the sample cuvettes. The lethal doses were calculated for each strain in order to estimate LD50
, and LD100
UV dosages. LD100
was defined as the UV dose at which no cultivable spores were recovered using the procedures described here.
Spores of nine strains that exhibited the highest LD90 were diluted with sterile deionized water to obtain approximately 1 x 106 spores ml–1. Then 2-ml aliquots of the suspensions were placed in 3-ml Suprasil quartz cuvettes (path length, 10 mm) equipped with a micro stir bar (3 by 3 mm; Fisher Scientific). When two strains were mixed for exposure, 5 x 105 spores ml–1 of each strain were mixed to obtain a final density of 106 spores ml–1. The UV exposure times ranged from 30 s to 30 min. At various intervals, 100-µl samples were removed, diluted serially 10-fold in sterile phosphate-buffered saline, and plated onto TSA. All TSA plates were incubated at 32°C for 24 to 48 h, and CFU were enumerated. The most resistant spores were selected for further experimentation. Quartz cuvettes were cleaned after each exposure experiment by rinsing them three times with 70% ethanol and then three times with 95% ethanol. The rinsed and dried cuvettes were placed in appropriate gas-permeable envelopes (Tyvek pouches; Advanced Sterilization Products) and sterilized by exposure to one to four cycles of hydrogen peroxide injection 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 (46). Various bandwidths were generated as described above. In all UV assays in which we 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 they were growing on agar medium.