Surface Characteristics of Spacecraft Components Affect the Aggregation of Microorganisms and May Lead to Different Survival Rates of Bacteria on Mars Landers

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Escherichia coli and Serratia liquefaciens, two bacterial spacecraft contaminants known to replicate under low atmospheric pressures of 2.5 kPa, were tested for growth and survival under simulated Mars conditions. Environmental stresses of high salinity, low temperature, and low pressure were screened alone and in combination for effects on bacterial survival and replication, and then cells were tested in Mars analog soils under simulated Mars conditions. Survival and replication of E. coli and S. liquefaciens cells in liquid medium were evaluated for 7 days under low temperatures (5, 10, 20, or 30°C) with increasing concentrations (0, 5, 10, or 20%) of three salts (MgCl 2 , MgSO 4 , NaCl) reported to be present on the surface of Mars. Moderate to high growth rates were observed for E. coli and S. liquefaciens at 30 or 20°C and in solutions with 0 or 5% salts. In contrast, cell densities of both species generally did not increase above initial inoculum levels under the highest salt concentrations (10 and 20%) and the four temperatures tested, with the exception that moderately higher cell densities were observed for both species at 10% MgSO 4 maintained at 20 or 30°C. Growth rates of E. coli and S. liquefaciens in low salt concentrations were robust under all pressures (2.5, 10, or 101.3 kPa), exhibiting a general increase of up to 2.5 orders of magnitude above the initial inoculum levels of the assays. Vegetative E. coli cells were maintained in a Mars analog soil for 7 days under simulated Mars conditions that included temperatures between 20 and ؊50°C for a day/night diurnal period, UVC irradiation (200 to 280 nm) at 3.6 W m ؊2 for daytime operations (8 h), pressures held at a constant 0.71 kPa, and a gas composition that included the top five gases found in the martian atmosphere. Cell densities of E. coli failed to increase under simulated Mars conditions, and survival was reduced 1 to 2 orders of magnitude by the interactive effects of desiccation, UV irradiation, high salinity, and low pressure (in decreasing order of importance). Results suggest that E. coli may be able to survive, but not grow, in surficial soils on Mars.The search for extant life on Mars remains a stated goal of NASA's Mars Exploration Program and Astrobiology Institutes (13,17). Intrinsic within such a life detection strategy is a requirement to understand how terrestrial life might survive, replicate, and proliferate on Mars. To mitigate the risks of the forward contamination of Mars, the bioloads on spacecrafts targeted for landing must be reduced to low density and diversity (4, 7). Planetary protection guidelines are designed to prevent both the forward contamination of the martian surface and to ensure the scientific integrity of any deployed life detection experiments. To date, 12 spacecraft have landed or crashed onto the Mars surface as a result of U.S., Russian, and European space program missions, but it is currently unknown if terrestrial microorganisms typically found on spacecraft surfaces can grow and replicate under con...

Section: Discussionsupporting

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Effects of Simulated Mars Conditions on the Survival and Growth of Escherichia coli and Serratia liquefaciens

Berry

Jenkins

Schuerger

2010

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“…Vectran fabric types A and B proved to be even more problematic for recovery of spores, with only 5.9% and 8.8% recovery efficiencies. Differences in surface textural and physiochemical adhesive properties may be the reason for significant variations in the recovery efficiencies of the different surfaces (35) and, in particular, of Vectran fabric types. On the other hand, a recent publication demonstrated that different seeding methods for spores (dry or liquid deposition) can significantly affect sam-pling success (13).…”

Section: Discussionmentioning

confidence: 99%

Validation of a Nylon-Flocked-Swab Protocol for Efficient Recovery of Bacterial Spores from Smooth and Rough Surfaces

Probst

Facius

Wirth

et al. 2010

In order to meet planetary-protection requirements, culturable bacterial spore loads are measured representatively for the total microbial contamination of spacecraft. However, the National Aeronautics and Space Administration's (NASA's) cotton swab protocols for spore load determination have not changed for decades. To determine whether a more efficient alternative was available, a novel swab was evaluated for recovery of different Bacillus atrophaeus spore concentrations on stainless steel and other surfaces. Two protocols for the nylon-flocked swab (NFS) were validated and compared to the present NASA standard protocol. The results indicate that the novel swab protocols recover 3-to 4-fold more (45.4% and 49.0% recovery efficiency) B. atrophaeus spores than the NASA standard method (13.2%). Moreover, the nylon-flocked-swab protocols were superior in recovery efficiency for spores of seven different Bacillus species, including Bacillus anthracis Sterne (recovery efficiency, 20%). The recovery efficiencies for B. atrophaeus spores from different surfaces showed a variation from 5.9 to 62.0%, depending on the roughness of the surface analyzed. Direct inoculation of the swab resulted in a recovery rate of about 80%, consistent with the results of scanning electron micrographs that allowed detailed comparisons of the two swab types. The results of this investigation will significantly contribute to the cleanliness control of future life detection missions and will provide significant improvement in detection of B. anthracis contamination for law enforcement and security efforts.

“…For this reason, Mars environmental simulation experiments have been conducted to estimate (i) the survival rates of terrestrial microorganisms and (ii) the persistence of organic molecules on Mars (3,4,10,15,25,29,30,37,38,45). Historically, several studies have explored the resistance of bacterial spores to simulated Martian conditions (8,10,12,14, and references therein); these studies have concentrated mainly on the survival of spores of wild-type strains of various spore-forming species (10,32,(38)(39)(40). More recent experiments have attempted to better understand the molecular factors causing spore resistance to environmental extremes, in which mainly spores of the model organism Bacillus subtilis that carry mutations affecting spore protective factors or spore DNA repair systems have been used (9, 18-23; reviewed in references 16, 28, 41, and 42).…”

mentioning

confidence: 99%

“…Triplicate air-dried spore samples with a thickness of approximately 25 spore layers (for the 5 ϫ 10 8 spore concentration) on presterilized spacecraftqualified, chemfilm-treated aluminum 6061 coupons (13 mm in diameter by 1 mm thick) (23) were exposed to simulated Martian conditions in a cylindrical Mars simulation chamber (MSC) as described in detail previously (37). The simulated Mars environmental conditions listed in Table 2 (38)(39)(40). Spore recovery from coupons was accomplished by stripping off spores with polyvinyl alcohol, and viability assays were performed as described previously (18,19,21,22).…”

mentioning

confidence: 99%

Protective Role of Spore Structural Components in Determining Bacillus subtilis Spore Resistance to Simulated Mars Surface Conditions

Moeller

Schuerger

Reitz

et al. 2012

Self Cite

Spores of wild-type and mutant Bacillus subtilis strains lacking various structural components were exposed to simulated Martian atmospheric and UV irradiation conditions. Spore survival and mutagenesis were strongly dependent on the functionality of all of the structural components, with small acid-soluble spore proteins, coat layers, and dipicolinic acid as key protectants.O ne major aspect of space biological research is the investigation of the responses of bacterial, viral, archaeal, and fungal species, as well as biomolecules, to simulated Martian conditions and their evaluation as potential forward contamination risks in the context of planetary protection (2, 4-7, 11, 12, 24, 30, 37-40, 44-46). For this reason, Mars environmental simulation experiments have been conducted to estimate (i) the survival rates of terrestrial microorganisms and (ii) the persistence of organic molecules on Mars (3,4,10,15,25,29,30,37,38,45). Historically, several studies have explored the resistance of bacterial spores to simulated Martian conditions (8,10,12,14, and references therein); these studies have concentrated mainly on the survival of spores of wild-type strains of various spore-forming species (10,32,(38)(39)(40). More recent experiments have attempted to better understand the molecular factors causing spore resistance to environmental extremes, in which mainly spores of the model organism Bacillus subtilis that carry mutations affecting spore protective factors or spore DNA repair systems have been used (9, 18-23; reviewed in references 16, 28, 41, and 42). Spores of B. subtilis possess brownish pigmentation, thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents such as the calcium chelate of dipicolinic acid (Ca-DPA) and ␣/␤-type small, acid-soluble spore proteins (SASP), all factors which have been previously found to contribute to spore resistance (reviewed in references 16, 28, 41, and 42). However, the possible roles of these factors in spore resistance to the extreme environmental conditions prevailing on the surface of Mars have not been explored, which is the subject of the present work.The B. subtilis strains used in this study are listed in Table 1, and all of the strains used were congenic to their respective wild-type strains. Spores were obtained by cultivation under vigorous aeration in double-strength liquid Schaeffer sporulation medium (36) under identical conditions for each strain, purified, and stored as described previously (31). When appropriate, chloramphenicol (5 g/ml), erythromycin (1 g/ml), spectinomycin (100 g/ml), or tetracycline (10 g/ml) was added to the medium. Spore preparations consisted of single spores with no detectable clumps and were Ͼ99% free of growing cells, germinated spores, and cell debris, as seen in a phase-contrast microscope. Triplicate air-dried spore samples with a thickness of approximately 25 spore layers (for the 5 ϫ 10 8 spore concentration) on presterilized sp...