Relationships between the use of Embden Meyerhof pathway (EMP) or Phosphoketolase pathway (PKP) and lactate production capabilities of diverse Lactobacillus reuteri strains.

The aims of this study is to compare the growth and glucose metabolism of three Lactobacillus reuteri strains (i.e. DSM 20016, DSM 17938, and ATCC 53608) which are lactic acid bacteria of interest used for diverse applications such as probiotics implying the production of biomass, or for the production of valuable chemicals (3-hydroxypropionaldehyde, 3-hydroxypropionic acid, 1,3-propanediol). However, the physiological diversity inside the species, even for basic metabolisms, like its capacity of acidification or glucose metabolism, has not been studied yet. In the present work, the growth and metabolism of three strains representative of the species diversity have been studied in batch mode. The strains were compared through characterization of growth kinetics and evaluation of acidification kinetics, substrate consumption and product formation. The results showed significant differences between the three strains which may be explained, at least in part, by variations in the distribution of carbon source between two glycolytic pathways during the bacterial growth: the phosphoketolase or heterolactic pathway (PKP) and the Embden-Meyerhof pathway (EMP). It was also shown that, in the context of obtaining a large amount of biomass, DSM 20016 and DSM 17938 strains were the most effective in terms of growth kinetics. The DSM 17938 strain, which shows the more significant metabolic shift from EMP to PKP when the pH decreases, is more effective for lactate production.

Pressure treatment of Saccharomyces cerevisiae in low-moisture environments.

We investigated the influence of cell hydration on the ability of Saccharomyces cerevisiae CBS 1171 to withstand extreme hydrostatic pressure in order to determine the mechanisms involved in cell resistance. Hydration conditions were modified in two different ways. We first modulated the chemical potential of water by adding glycerol in cell suspensions. Another procedure consisted in dehydrating cells aerobically and immersing them in perfluorooctane, an innocuous hydrophobic liquid used as a pressure-transmitting medium, prior to pressure treatments. This original method made it possible to transmit isostatic pressure to yeast powders without changing the initial water activity (aw) level at which cells had been equilibrated. The aw ranged between 0.11 and 0.99. Pressure treatments were applied at levels of up to 600 MPa for 10 min, 24 h, and 6 days. The dehydration of cells was found to strongly limit, or even prevent, cell inactivation under pressure. Notably, cells suspended in a water-glycerol mixture with aw levels of 0.71 or below were completely protected against all pressure treatments. Moreover, cells dehydrated aerobically survived for 6 days at 600 MPa even when aw levels were relatively high (up to 0.94). We highlighted the crucial role of water content in determining cellular damage under pressure. When water is available in a sufficient amount, high pressure induces membrane permeabilization, causing uncontrolled mass transfers that could lead to death during a prolonged holding under pressure. Possible mechanisms of membrane permeabilization are discussed.

Cell inactivation and membrane damage after long-term treatments at sub-zero temperature in the supercooled and frozen states.

The survival of cells subjected to cooling at sub-zero temperature is of paramount concern in cryobiology. The susceptibility of cells to cryopreservation processes, especially freeze-thawing, stimulated considerable interest in better understanding the mechanisms leading to cell injury and inactivation. In this study, we assessed the viability of cells subjected to cold stress, through long-term supercooling experiments, versus freeze-thawing stress. The viability of Escherichia coli, Saccharomyces cerevisiae, and leukemia cells were assessed over time. Supercooled conditions were maintained for 71 days at -10 degrees C, and for 4 h at -15 degrees C, and -20 degrees C, without additives or emulsification. Results showed that cells could be inactivated by the only action of sub-zero temperature, that is, without any water crystallization. The loss of cell viability upon exposure to sub-zero temperatures is suggested to be caused by exposure to cold shock which induced membrane damage. During holding time in the supercooled state, elevated membrane permeability results in uncontrolled mass transfer to and from the cell maintained at cold conditions and thus leads to a loss of viability. With water crystallization, cells shrink suddenly and thus are exposed to cold osmotic shock, which is suggested to induce abrupt loss of cell viability. During holding time in the frozen state, cells remain suspended in the residual unfrozen fraction of the liquid and are exposed to cold stress that would cause membrane damage and loss of viability over time. However, the severity of such a stress seems to be moderated by the cell type and the increased solute concentration in the unfrozen fraction of the cell suspension.

Damage in Escherichia coli cells treated with a combination of high hydrostatic pressure and subzero temperature.

The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the cell suspension in situ under high hydrostatic pressure (HHP) and subzero-temperature conditions made it possible to show the partial reversibility of pressure-induced nucleoid condensation. However, based on visual examination of TEM micrographs, protein aggregation did not seem to be reversible. Reversible cell membrane permeabilization was noticeable, particularly for HHP treatments at subzero temperature. A correlation between membrane permeabilization and cell inactivation was established, suggesting different mechanisms at room and subzero temperatures. We propose that the inactivation of E. coli cells under combined HHP and subzero temperature occurs mainly during their transiently permeabilized state, whereas HHP inactivation at room temperature is related to a balance of transient and permanent permeabilization. The correlation between TEM results and cell inactivation was not absolute. Further work is required to elucidate the effects of pressure-induced damage on nucleoids and proteins during cell inactivation.

Synergistic and antagonistic effects of combined subzero temperature and high pressure on inactivation of Escherichia coli.

The combined effects of subzero temperature and high pressure on the inactivation of Escherichia coli K12TG1 were investigated. Cells of this bacterial strain were exposed to high pressure (50 to 450 MPa, 10-min holding time) at two temperatures (-20 degrees C without freezing and 25 degrees C) and three water activity levels (a(w)) (0.850, 0.992, and ca. 1.000) achieved with the addition of glycerol. There was a synergistic interaction between subzero temperature and high pressure in their effects on microbial inactivation. Indeed, to achieve the same inactivation rate, the pressures required at -20 degrees C (in the liquid state) were more than 100 MPa less than those required at 25 degrees C, at pressures in the range of 100 to 300 MPa with an a(w) of 0.992. However, at pressures greater than 300 MPa, this trend was reversed, and subzero temperature counteracted the inactivation effect of pressure. When the amount of water in the bacterial suspension was increased, the synergistic effect was enhanced. Conversely, when the a(w) was decreased by the addition of solute to the bacterial suspension, the baroprotective effect of subzero temperature increased sharply. These results support the argument that water compression is involved in the antimicrobial effect of high pressure. From a thermodynamic point of view, the mechanical energy transferred to the cell during the pressure treatment can be characterized by the change in volume of the system. The amount of mechanical energy transferred to the cell system is strongly related to cell compressibility, which depends on the water quantity in the cytoplasm.