Role of growth phase and ethanol in freeze-thaw stress resistance of Saccharomyces cerevisiae

Abstract

The freeze-thaw tolerance of Saccharomyces cerevisiae was examined throughout growth in aerobic batch culture. Minimum tolerance to rapid freezing (immersion in liquid nitrogen; cooling rate, approximately 200'C min-') was associated with respirofermentative (exponential) growth on glucose. However, maximum tolerance occurred not during the stationary phase but during active respiratory growth on ethanol accumulated during respirofermentative growth on glucose. The peak in tolerance occurred several hours after entry into the respiratory growth phase and did not correspond to a transient accumulation of trehalose which occurred at the point of glucose exhaustion. Substitution of ethanol with other carbon sources which permit high levels of respiration (acetate and galactose) also induced high freeze-thaw tolerance, and the peak did not occur in cells shifted directly from fermentative growth to starvation conditions or in two respiratorily incompetent mutants. These results imply a direct link with respiration, rather than exhaustion of glucose. The role of ethanol as a cryoprotectant per se was also investigated, and under conditions of rapid freezing (cooling rate, approximately 200°C min-'), ethanol demonstrated a significant cryoprotective effect. Under the same freezing conditions, glycerol had little effect at high concentrations and acted as a cryosensitizer at low concentrations. Conversely, under slow-freezing conditions (step freezing at -20, -70, and then -196°C; initial cooling rate, approximately 3C min-'), glycerol acted as a cryoprotectant while ethanol lost this ability.Ethanol may thus have two effects on the cryotolerance of baker's yeast, as a respirable carbon source and as a cryoprotectant under rapid-freezing conditions.

Stress tolerance in a yeast sterol auxotroph: role of ergosterol, heat shock proteins and trehalose

“…For trehalose determination cells were harvested from 80 ml of culture and washed twice with chilled distilled water. Trehalose was extracted with cold 0.5 M trichloroacetic acid and estimated according to a modi¢ed anthrone procedure as previously described [14]. The content of trehalose was expressed as a percentage of the dry weight of cells.…”

Cryopreservation of competent intact yeast cells for efficient electroporation

“…The cell‐growth stage of yeast is an important factor in both cryopreservation and transformation. Yeast cells in the stationary phase generally become more resistant to cryoinjury and hypertonic stress than do those in the exponential phase (Morris et al ., 1988; Lewis et al ., 1993). It has been reported that compatible solutes such as glycerol and trehalose were transiently accumulated during the stationery phase (Gélinas et al ., 1989; Lewis et al ., 1993).…”

“…For the reasons mentioned above, the membrane water permeability responsible for thawing injuries is also an important factor in cryopreservation of competent cell for electroporation. The water permeability differs from strain to strain and is reflected by growth conditions such as growth phase and growth medium (Lewis et al ., 1993; Gélinas et al ., 1991). Therefore, transformation efficiency after cryopreservation would be influenced by the membrane permeability based on the strain and growth conditions.…”

Improvement in lactone production from biotransformation of ricinoleic acid based on the porous starch delivery system

“…It has been reported that the exogenous glycerol was a less toxic cryoprotectant for yeast cells and enzyme. 38 The glycerol could affect lipid composition of the cell membrane by passive diffusion into the cell which could enhance the tolerance of the yeast to environment stress. 39,40 When the glycerol gets into the yeast, it could act as a carbon source which is metabolically phosphorylated to glycerol-3-P to form dihydroxyacetone-3-P after the oxidation.…”