Surface characteristics of spacecraft components affect the aggregation of microorganisms and may lead to different survival rates of bacteria on Mars landers.

Layers of dormant endospores of Bacillus subtilis HA101 were applied to eight different spacecraft materials and exposed to martian conditions of low pressure (8.5 mbar), low temperature (-10 degrees C), and high CO(2) gas composition and irradiated with a Mars-normal ultraviolet (UV-visible- near-infrared spectrum. Bacterial layers were exposed to either 1 min or 1 h of Mars-normal UV irradiation, which simulated clear-sky conditions on equatorial Mars (0.1 tau). When exposed to 1 min of Mars UV irradiation, the numbers of viable endospores of B. subtilis were reduced three to four orders of magnitude for two brands of aluminum (Al), stainless steel, chemfilm-treated Al, clear-anodized Al, and black-anodized Al coupons. In contrast, bacterial survival was reduced only one to two orders of magnitude for endospores on the non-metal materials astroquartz and graphite composite when bacterial endospores were exposed to 1 min of Mars UV irradiation. When bacterial monolayers were exposed to 1 h of Mars UV irradiation, no viable bacteria were recovered from the six metal coupons listed above. In contrast, bacterial survival was reduced only two to three orders of magnitude for spore layers on astroquartz and graphite composite exposed to 1 h of Mars UV irradiation. Scanning electron microscopy images of the bacterial monolayers on all eight spacecraft materials revealed that endospores of B. subtilis formed large aggregates of multilayered spores on astroquartz and graphite composite, but not on the other six spacecraft materials. It is likely that the formation of multilayered aggregates of endospores on astroquartz and graphite composite is responsible for the enhanced survival of bacterial cells on these materials.

Recurrent isolation of hydrogen peroxide-resistant spores of Bacillus pumilus from a spacecraft assembly facility.

While the microbial diversity of a spacecraft assembly facility at the Jet Propulsion Laboratory (Pasadena, CA) was being monitored, H2O2-resistant bacterial strains were repeatedly isolated from various surface locations. H2O2 is a possible sterilant for spacecraft hardware because it is a low-temperature process and compatible with various modern-day spacecraft materials, electronics, and components. Both conventional biochemical testing and molecular analyses identified these strains as Bacillus pumilus. This Bacillus species was found in both unclassified (entrance floors, anteroom, and air-lock) and classified (floors, cabinet tops, and air) locations. Both vegetative cells and spores of several B. pumilus isolates were exposed to 5% liquid H2O2 for 60 min. Spores of each strain exhibited higher resistance than their respective vegetative cells to liquid H2O2. Results indicate that the H2O2 resistance observed in both vegetative cells and spores is strain-specific, as certain B. pumilus strains were two to three times more resistant than a standard Bacillus subtilis dosimetry strain. An example of this trend was observed when the type strain of B. pumilus, ATCC 7061, proved sensitive, whereas several environmental strains exhibited varying degrees of resistance, to H2O2. Repeated isolation of H2O2-resistant strains of B. pumilus in a clean-room is a concern because their persistence might potentially compromise life-detection missions, which have very strict cleanliness and sterility requirements for spacecraft hardware.

Evaluation of various cleaning methods to remove bacillus spores from spacecraft hardware materials.

A detailed study was made of the biological cleaning effectiveness, defined in terms of the ability to remove bacterial spores, of a number of methods used to clean hardware surfaces. Aluminum (Al 6061) and titanium (Ti 6Al-4V) were chosen for the study as they were deemed the two materials most likely to be used in spacecraft extraterrestrial sampler construction. Metal coupons (1 cm x 2.5 cm) were precleaned and inoculated with 5.8 x 10(3) cultivable Bacillus subtilis spores, which are commonly found on spacecraft surfaces and in the assembly environments. The inoculated coupons were subsequently cleaned using: (1) 70% isopropyl alcohol wipe; (2) water wipe; (3) multiple-solvent flight-hardware cleaning procedures used at the Jet Propulsion Laboratory (JPL); (4) Johnson Space Center-developed ultrapure water rinse; and (5) a commercial, semi-aqueous, multiple-solvent (SAMS) cleaning process. The biological cleaning effectiveness was measured by agar plate assay, sterility test (growing in liquid media), and epifluorescent microscopy. None of the cleaning protocols tested completely removed viable spores from the surface of the aluminum. In contrast, titanium was capable of being cleaned to sterility by two methods, the JPL standard and the commercial SAMS cleaning process. Further investigation showed that the passivation step employed in the JPL standard method is an effective surface sterilant on both metals but not compatible with aluminum. It is recommended that titanium (Ti 6Al-4V) be considered superior to aluminum (Al 6061) for use in spacecraft sampling hardware, both for its potential to be cleaned to sterilization and for its ability to withstand the most effective cleaning protocols.

Survival of endospores of Bacillus subtilis on spacecraft surfaces under simulated martian environments: implications for the forward contamination of Mars.

Experiments were conducted in a Mars simulation chamber (MSC) to characterize the survival of endospores of Bacillus subtilis under high UV irradiation and simulated martian conditions. The MSC was used to create Mars surface environments in which pressure (8.5 mb), temperature (-80, -40, -10, or +23 degrees C), gas composition (Earth-normal N2/O2 mix, pure N2, pure CO2, or a Mars gas mix), and UV-VIS-NIR fluence rates (200-1200 nm) were maintained within tight limits. The Mars gas mix was composed of CO2 (95.3%), N2 (2.7%), Ar (1.7%), O2 (0.2%), and water vapor (0.03%). Experiments were conducted to measure the effects of pressure, gas composition, and temperature alone or in combination with Mars-normal UV-VIS-NIR light environments. Endospores of B. subtilis, were deposited on aluminum coupons as monolayers in which the average density applied to coupons was 2.47 x 10(6) bacteria per sample. Populations of B. subtilis placed on aluminum coupons and subjected to an Earth-normal temperature (23 degrees C), pressure (1013 mb), and gas mix (normal N2/O2 ratio) but illuminated with a Mars-normal UV-VIS-NIR spectrum were reduced by over 99.9% after 30 sec exposure to Mars-normal UV fluence rates. However, it required at least 15 min of Mars-normal UV exposure to reduce bacterial populations on aluminum coupons to non-recoverable levels. These results were duplicated when bacteria were exposed to Mars-normal environments of temperature (-10 degrees C), pressure (8.5 mb), gas composition (pure CO2), and UV fluence rates. In other experiments, results indicated that the gas composition of the atmosphere and the temperature of the bacterial monolayers at the time of Mars UV exposure had no effects on the survival of bacterial endospores. But Mars-normal pressures (8.5 mb) were found to reduce survival by approximately 20-35% compared to Earth-normal pressures (1013 mb). The primary implications of these results are (a) that greater than 99.9% of bacterial populations on sun-exposed surfaces of spacecraft are likely to be inactivated within a few tens of seconds to a few minutes on the surface of Mars, and (b) that within a single Mars day under clear-sky conditions bacterial populations on sun-exposed surfaces of spacecraft will be sterilized. Furthermore, these results suggest that the high UV fluence rates on the martian surface can be an important resource in minimizing the forward contamination of Mars.

Microbial characterization of the Mars Odyssey spacecraft and its encapsulation facility.

Microbial characterization of the Mars Odyssey spacecraft and the Kennedy Space Center Spacecraft Assembly and Encapsulation Facility II (SAEF-II) was carried out by both culture-based and molecular methods. The most dominant cultivable microbes were species of Bacillus, with comamonads, microbacteria and actinomycetales also represented. Several spore-forming isolates were resistant to gamma-radiation, UV, H2O2 and desiccation, and one Acinetobacter radioresistens isolate and several Aureobasidium, isolated directly from the spacecraft, survived various conditions. Sequences arising in clone libraries were fairly consistent between the spacecraft and facility; predominant genera included Variovorax, Ralstonia and Aquaspirillum. This study improves our understanding of the microbial community structure, diversity and survival capabilities of microbes in an encapsulation facility and physically associated with colocated spacecraft.

Bacillus nealsonii sp. nov., isolated from a spacecraft-assembly facility, whose spores are gamma-radiation resistant.

One of the spore-formers isolated from a spacecraft-assembly facility, belonging to the genus Bacillus, is described on the basis of phenotypic characterization, 16S rDNA sequence analysis and DNA-DNA hybridization studies. It is a Gram-positive, facultatively anaerobic, rod-shaped eubacterium that produces endospores. The spores of this novel bacterial species exhibited resistance to UV, gamma-radiation, H2O2 and desiccation. The 18S rDNA sequence analysis revealed a clear affiliation between this strain and members of the low G+C Firmicutes. High 16S rDNA sequence similarity values were found with members of the genus Bacillus and this was supported by fatty acid profiles. The 16S rDNA sequence similarity between strain FO-92T and Bacillus benzoevorans DSM 5391T was very high. However, molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in this genus, but DNA-DNA hybridization data support the proposal of FO-92T as Bacillus nealsonii sp. nov. (type strain is FO-92T =ATCC BAAM-519T =DSM 15077T).

ATP as a biomarker of viable microorganisms in clean-room facilities.

A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.