Use of NanoSIMS to Identify the Lower Limits of Metabolic Activity and Growth by Serratia liquefaciens Exposed to Sub-Zero Temperatures.

Serratia liquefaciens is a cold-adapted facultative anaerobic astrobiology model organism with the ability to grow at a Martian atmospheric pressure of 7 hPa. Currently there is a lack of data on its limits of growth and metabolic activity at sub-zero temperatures found in potential habitable regions on Mars. Growth curves and nano-scale secondary ion mass spectrometry (NanoSIMS) were used to characterize the growth and metabolic threshold for S. liquefaciens ATCC 27,592 grown at and below 0 °C. Cells were incubated in Spizizen medium containing three stable isotopes substituting their unlabeled counterparts; i.e., 13C-glucose, (15NH4)2SO4, and H218O; at 0, -1.5, -3, -5, -10, or -15 °C. The isotopic ratios of 13C/12C, 15N/14N, and 18O/16O and their corresponding fractions were determined for 240 cells. NanoSIMS results revealed that with decreasing temperature the cellular amounts of labeled ions decreased indicating slower metabolic rates for isotope uptake and incorporation. Metabolism was significantly reduced at -1.5 and -3 °C, almost halted at -5 °C, and shut-down completely at or below -10 °C. While growth was observed at 0 °C after 5 days, samples incubated at -1.5 and -3 °C exhibited significantly slower growth rates until growth was detected at 70 days. In contrast, cell densities decreased by at least half an order of magnitude over 70 days in cultures incubated at ≤ -5 °C. Results suggest that S. liquefaciens, if transported to Mars, might be able to metabolize and grow in shallow sub-surface niches at temperatures above -5 °C and might survive-but not grow-at temperatures below -5 °C.