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

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 (MgCl₂, MgSO₄, 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% MgSO₄ 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 conditions encountered on the surface (44, 45, 48).Despite cleaning and sterilization measures taken to significantly reduce microbial bioloads on spacecraft (26, 56), diverse microbial communities remain at the time of launch (7, 31, 32, 44). The diversity of microorganisms found on spacecraft surfaces are generally characteristic of the clean rooms within which the spacecraft are processed. Spacecraft assembly facilities are oligotrophic extreme environments in which only the most resilient species survive the high-desiccation, low-nutrient conditions, controlled air circulation, and the rigors of bioburden reduction (56, 57). The biological inventory of microorganisms on spacecraft has mostly been limited to isolation and identification using standard culture-based microbiological assays (44, 48, 53). However, culture-based microbiological assays likely underestimate the biological diversity present on spacecraft, as traditional culture techniques fail to capture more than 99.9% of present phylotypes (7). Recently, the simultaneous use of culture-dependent and culture-independent techniques (e.g., Limulus amoebocyte lysate assay [LAL], ATP bioluminescence assay, lipopolysaccharide-based microbial detection, and DNA-based PCR) have identified many nonculturable species (31, 32, 57). Known culturable bacteria recovered from spacecraft surfaces include, but are not limited to, species of Acinetobacter, Bacillus, Corynebacterium, Escherichia, Flavobacterium, Micrococcus, Pseudomonas, Serratia, Staphylococcus, and Streptococcus (44, 53, 57).After launch, spacecraft are exposed to interplanetary conditions of ultralow pressure (3 × 10⁻¹⁰ kPa), extreme desiccating conditions, fluctuating temperatures, solar UV irradiation, and ionizing radiation (22, 44). Furthermore, upon landing, the conditions on the surface of Mars are not much improved over interplanetary space. Diverse biocidal or inhibitory conditions on Mars have been identified in a number of recent publications (8, 21, 22, 35, 36, 38, 44, 48, 59) and include the following (not in order of priority): solar UVC irradiation, low pressure, extreme desiccating conditions, extreme diurnal temperature fluctuations, solar particle events, galactic cosmic rays, UV glow discharge from blowing dust, solar UV-induced volatile oxidants (e.g., O₂⁻, O⁻, H₂O₂, NO_x, O₃), globally distributed oxidizing soils, extremely high salt levels (e.g., MgCl₂, NaCl, FeSO₄, and MgSO₄) in surficial soils at some sites on Mars, high concentrations of heavy metals in martian soils, acidic conditions in martian regolith, high CO₂ concentrations in the global atmosphere, and presence of perchlorates in some regoliths. UV irradiation, especially UVC photons (200 to 280 nm), may be the most biocidal of all factors to microbial survival on the martian surface (34, 37, 39, 47, 50, 52). Microorganisms found on sun-exposed surfaces of spacecraft are killed off within a few tens of minutes of exposure; but if covered by as little as a few hundred micrometers of martian soil, significant protection is provided (11, 34, 47). It is currently unknown if terrestrial microorganisms typically found on spacecraft surfaces can grow and replicate under conditions encountered on the surface of Mars (44, 48).In the studies cited above, most research focused on the survival of dormant spores or vegetative cells under Mars conditions. In contrast, only a few papers have explored the possibility of growth and replication of terrestrial microorganisms under environmental conditions that approach those found in surficial soils of Mars (5, 25, 45, 48). Of these four, 2.5 kPa is the lowest pressure at which replication was observed for a few bacterial species (5, 45, 48).The primary objective of the current study was to expose two non-spore-forming species to environmental stresses present on the surface of Mars to characterize the potential response of the bacteria to martian temperatures, salinities, and pressures. Two bacterial species, Escherichia coli and Serratia liquefaciens, were selected from over 30 prokaryotic species tested in preliminary experiments (5, 45). Their selection was based on their common association with humans, recovery from robotic spacecraft and space-based human life support systems (44, 53), and demonstrated replication at 2.5 kPa of total atmospheric pressure (5, 45). Experiments were conducted on cell suspensions in liquid medium at combinations of low pressure, high salt concentrations, and low temperatures, and then with cells mixed into soils and exposed to simulated Mars conditions. It was predicted for cell suspensions that (i) low temperatures would dramatically retard cell proliferation, (ii) high concentrations of salts would be biocidal on cell suspensions, and (iii) low pressure would have weak to moderate inhibitory effects on cell growth of both species. For cells in soils, growth was not expected under Mars simulations which exposed vegetative cells to low pressure, low temperatures, anaerobic gas composition, and high UVC irradiation similar to the martian surface. Although replication was not predicted, bacterial survival in analog Mars soils under simulated Mars conditions was anticipated.