Effects of simulated Mars conditions on the survival and growth of Escherichia coli and Serratia liquefaciens

Abstract

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 degrees 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 degrees 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 degrees 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 degrees 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.

FIG. 1.

Culture growth of Escherichia coli and Serratia liquefaciens in mLB broth maintained at 5, 10, 20, or 30°C. Cultures were incubated for 7 days in CO₂ atmospheres. Error bars denote 95% confidence intervals (n = 6).

FIG. 2.

Culture growth of Escherichia coli in mLB broth doped with 5, 10, or 20% MgSO₄, MgCl₂, or NaCl. Cultures were incubated for 7 days in CO₂ atmospheres and maintained at 5, 10, 20, or 30°C. Error bars denote 95% confidence intervals (n = 6).

FIG. 3.

Culture growth of Serratia liquefaciens in mLB broth doped with 5, 10, or 20% MgSO₄, MgCl₂, or NaCl. Cultures were incubated for 7 days in CO₂ atmospheres and maintained at 5, 10, 20, or 30°C. Error bars denote 95% confidence intervals (n = 6).

FIG. 4.

Survival of Escherichia coli and Serratia liquefaciens after 7 days under low temperatures and high salinity in CO₂ atmospheres. Only treatments which resulted in no increase in OD in the first experiment (Fig. 2 and 3) were included in the new assay. Cultures were harvested and counted after 7 days of incubation at 5, 10, 20, or 30°C. Dashed lines indicate starting levels of inoculum. Error bars denote 95% confidence intervals (n = 6).

FIG. 5.

Viable cell densities of Escherichia coli and Serratia liquefaciens with and without salts under low pressure in CO₂ atmospheres. Cultures were incubated in mLB at 20°C for 7 days under 2.5, 10.0, or 101.3 kPa. Dashed lines indicate starting levels of inoculum. Error bars denote 95% confidence intervals (n = 6).

FIG. 6.

Viable cell densities of Escherichia coli and Serratia liquefaciens recovered from Mars analog soils after desiccation (Des.) or incubation at 20, 4, or −20°C for 1 or 7 days. Dashed lines indicate starting levels of inoculum. Error bars denote 95% confidence intervals (n = 9).

FIG. 7.

Recovery of viable Escherichia coli cells in Mars simulations and Earth controls. Escherichia coli cells were incubated for 7 days in Mars analog soils in Earth-normal conditions and in the MSC with and without UV exposure. Diurnal conditions within the MSC were changed from 20°C and UV irradiation (daytime) to −50°C without UV (nighttime). A Mars atmosphere was maintained at 0.71 kPa. The dashed line represents the average initial inoculum. Error bars denote 95% confidence intervals (n = 9).