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The effects of KCl, NaCl, and LiCl on the growth of Debaryomyces hansenii, usually considered a halotolerant yeast, and Saccharomyces cerevisiae were compared. KCl and NaCl had similar effects on D. hansenii, indicating that NaCl created only osmotic stress, while LiCl had a specific inhibitory effect, although relatively weaker than in S. cerevisiae. In media with low K ؉ , Na ؉ was able to substitute for K ؉ , restoring the specific growth rate and the final biomass of the culture. The intracellular concentration of Na ؉ reached values up to 800 mM, suggesting that metabolism is not affected by rather high concentrations of salt. The ability of D. hansenii to extrude Na ؉ and Li ؉ was similar to that described for S. cerevisiae, suggesting that this mechanism is not responsible for the increased halotolerance. Also, the kinetic parameters of Rb ؉ uptake in D. hansenii (V max , 4.2 nmol mg [dry weight] ؊1 min ؊1 ; K m , 7.4 mM) indicate that the transport system was not more efficient than in S. cerevisiae. Sodium (50 mM) activated the transport of Rb ؉ by increasing the affinity for the substrate in D. hansenii, while the effect was opposite in S. cerevisiae. Lithium inhibited Rb ؉ uptake in D. hansenii. We propose that the metabolism of D. hansenii is less sensitive to intracellular Na ؉ than is that of S. cerevisiae, that Na ؉ substitutes for K ؉ when K ؉ is scarce, and that the transport of K ؉ is favored by the presence of Na ؉. In low K ؉ environments, D. hansenii behaved as a halophilic yeast.

The effect of acetic acid on transport of glucose and on the activity of glycolytic enzymes of Saecharomyees eerevisiae was investigated. Acetic acid did not affect glucose transport. The inhibitory effect of the acid on the enzymes was considered from the point of view of acidification of the cytoplasm (pH dependence of the activity) and of the direct effect of the presence of acetic acid. Enolase was the enzyme most severely affected according to these two criteria. Fermentation was monitored in vivo by 31p-NMR. When ATP was available, a rise in cytoplasmic pH was observed and fermentation proceeded with a lower level of sugar phosphate. This may indicate that control was exerted at one of the early phosphorylation steps.

The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into the mechanisms involved in ethanol toxicity and tolerance in this organism. Exposure to ethanol resulted in an increase in the permeability of the cytoplasmic membrane, enhancing passive proton influx and concomitant loss of intracellular material (absorbing at 260 nm). Cells grown in the presence of 8% (vol/vol) ethanol revealed adaptation to ethanol stress, since these cells showed higher retention of compounds absorbing at 260 nm. Moreover, for concentrations higher than 10% (vol/vol), lower rates of passive proton influx were observed in these ethanol-adapted cells, especially at pH 3.5. The effect of ethanol on O. oeni cells was studied as the ability to efficiently retain carboxyfluorescein (cF) as an indicator of membrane integrity and enzyme activity and the uptake of propidium iodide (PI) to assess membrane damage. Flow cytometric analysis of both ethanol-adapted and nonadapted cells with a mixture of the two fluorescent dyes, cF and PI, revealed three main subpopulations of cells: cF-stained intact cells; cF-and PI-stained permeable cells, and PI-stained damaged cells. The subpopulation of O. oeni cells that maintained their membrane integrity, i.e., cells stained only with cF, was three times larger in the population grown in the presence of ethanol, reflecting the protective effect of ethanol adaptation. This information is of major importance in studies of microbial fermentations in order to assign bulk activities measured by classical methods to the very active cells that are effectively responsible for the observations.