Physiological changes induced in bacteria following pH stress as a model for space research

Abstract The physiology of the environmental bacterium Cupriavidus metallidurans CH34 (previously Ralstonia metallidurans) is being studied in comparison to the clinical model bacterium Escherichia coli in order to understand its behaviour and resistance under extreme conditions (pH, temperature, etc.). This knowledge is of importance in the light of the potential use and interest of this strain for space biology and bioremediation. Flow cytometry provides powerful means to measure a wide range of cell characteristics in microbiological research. In order to estimate physiological changes associated with pH stress, flow cytometry was employed to estimate the extent of damage on cell size, membrane integrity and potential, and production of superoxides in the two bacterial strains. Suspensions of C. metallidurans and E. coli were submitted to a 1-h pH stress (2 to 12). For flow cytometry, fluorochromes, including propidium iodide, 3,  3 ′ -dihexyloxacarbocyanine iodide and hydroethidine were chosen as analytical parameters for identifying the physiological state and the overall fitness of individual cells. A physiologic state of the bacterial population was assessed with a Coulter EPICS XL analyser based on the differential uptakes of these fluorescent stains. C. metallidurans cells exhibited a different staining intensity than E. coli cells. For both bacterial strains, the physiological status was only slightly affected between pH 6 and 8 in comparison with pH 7 which represents the reference pH. Moderate physiological damage could be observed at pH 4 and 5 as well as at pH 9 in both strains. At pH 2, 10 and 12, membrane permeability and potential and superoxide anion production were increased to high levels showing dramatic physiological changes. It is apparent that a range of significant physiological alterations occurs after pH stress. Fluorescent staining methods coupled with flow cytometry are useful and complementary for monitoring physiological changes induced not only by pH stress but also temperature and oxidative stress, radiation, pressure as well as space stress.