Graham G. Stewart

A Study of Ethanol Tolerance in Yeast

The ethanol tolerance of yeast and other microorganisms has remained a controversial area despite the many years of study. The complex inhibition mechanism of ethanol and the lack of a universally accepted definition and method to measure ethanol tolerance have been prime reasons for the controversy. A number of factors such as plasma membrane composition, media composition, mode of substrate feeding, osmotic pressure, temperature, intracellular ethanol accumulation, and byproduct formation have been shown to influence the ethanol tolerance of yeast. Media composition was found to have a profound effect upon the ability of a yeast strain to ferment concentrated substrates (high osmotic pressure) and to ferment at higher temperatures. Supplementation with peptone-yeast extract, magnesium, or potassium salts has a significant and positive effect upon overall fermentation rates. An intracellular accumulation of ethanol was observed during the early stages of fermentation. As fermentation proceeds, the intracellular and extracellular ethanol concentrations become similar. In addition, increases in osmotic pressure are associated with increased intracellular accumulation of ethanol. However, it was observed that nutrient limitation, not increased intracellular accumulation of ethanol, is responsible to some extent for the decreases in growth and fermentation activity of yeast cells at higher osmotic pressure and temperature.

Selection and optimization of yeast suitable for ethanol production at 40°C

Abstract A total of 65 yeast strains from various genera were screened for their ability to grow on glucose at 40°C for the purpose of isolating a thermotolerant yeast strain. Of these, only one strain, Saccharomyces diastaticus 62, was found capable of completely utilizing 15% glucose at this temperature, producing 6.4% (w/v) ethanol. When the glucose concentration was increased to 20%, to reflect more closely industrial practices, this strain could not completely utilize the sugar, producing 7.0% (w/v) ethanol with 4.5% glucose remaining in the medium. However, by doubling the nutrient components in the medium, complete glucose utilization was observed, resulting in the production of 9.1% (w/v) ethanol. Increasing the fermentation temperature from 40 to 45°C resulted in a decrease in the rate and extent of glucose utilization and ethanol production. The detrimental effect of increasing the temperature can be overcome by increasing the nutrient components in the medium. For example, at 43°C complete utilization of 15% glucose resulting in 6.8% (w/v) ethanol was observed only when the medium was supplemented with 2× PYN components. The role of magnesium in relieving the detrimental effect of high temperature may to some extent be related to the requirement of some of the glycolytic enzymes for this cation. In addition, increasing the cell density also resulted in an increase in ethanol production at the higher temperature. The best results were observed with cell concentrations in the range of 2–3.5% (w/v) wet weight.

The involvement of trehalose in yeast stress tolerance

SummaryA total of 12 yeast strains from various genera were examined for their ability to produce ethanol in the presence of high concentrations of glucose. From these studies, the yeastsTorulaspora delbrueckii andZygosaccharomyces rouxii were observed to the most osmotolerant. These osmotolerant yeast strains were also observed to possess high concentrations of intracellular trehalose. Futhermore, these strains were found to be tolerant to long-term storage at −20°C and to storage at 4°C in beer containing 5% (v/v) ethanol. Cells containing high trehalose levels at the time of freezing or cold storage exhibited the highest cell viabilities. Trehalose concentration was observed to increase during growth on glucose, reaching a maximum after 24–48 h. Increasing the incubation temperature from 21 to 40°C also resulted in an increase in intracellular trehalose content. These results suggest that trehalose plays a role in enhancing yeast survival under environmentally stressful conditions.

Sugar utilization by yeast during fermentation

SummaryWhen glucose and fructose are fermented separately, the uptake profiles indicate that both sugars are utilized at similar rates. However, when fermentations are conducted in media containing an equal concentration of glucose and fructose, glucose is utilized at approximately twice the rate of fructose. The preferential uptake of glucose also occurred when sucrose, which was first rapidly hydrolyzed into glucose and fructose by the action of the enzyme invertase, was employed as a substrate. Similar results were observed in the fermentation of brewers wort and wort containing 30% sucrose and 30% glucose as adjuncts. In addition, the high levels of glucose in the wort exerted severe catabolite repression on maltose utilization in theSaccharmyces uvarum (carlsbergensis) brewing strain. Kinetic analysis of glucose and fructose uptake inSaccharomyces cerevisiae revealed aKm of 1.6 mM for glucose and 20 mM for fructose. Thus, the yeast strain has a higher affinity for glucose than fructose. Growth on glucose or fructose had no repressible effect on the uptake of either sugar. In addition, glucose inhibited fructose uptake by 60% and likewise fructose inhibited, glucose uptake by 40%. These results indicate that glucose and fructose share the same membrane transport components.

Osmotic pressure effects and intracellular accumulation of ethanol in yeast during fermentation

SummaryThe intracellular accumulation of ethanol in yeast and its potential effects on growth and fermentation have been topics of controversy for the past several years. The determination of intracellular ethanol based on the exclusion of [14C]sorbitol to estimate aqueous cell volume was used to examine the question of intracellular ethanol accumulation. An intracellular accumulation of ethanol inSaccharomyces cerevisiae was observed during the early stages of fermentation. However, as fermentation continued, the intracellular and extracellular concentrations of ethanol became similar. Increasing the osmotic pressure of the medium with glucose or sorbitol was observed to cause an increase in the intracellular ethanol concentration. Associated with this was a decrease in yeast growth and fermentation rates. In addition, increasing the osmotic pressure of the medium was observed to cause an increase in glycerol production. Supplementation of the media with excess peptone, yeast extract, magnesium sulfate and potassium phosphate was found to relieve the detrimental effects of high osmotic pressure. Under these conditions, though, no effect on the intracellular and extracellular ethanol distribution was observed. These results indicate that nutrient limitation, and not necessarily intracellular ethanol accumulation, plays a key role during yeast fermentations in media of high osmolarity.

Alterations in fatty acid composition and trehalose concentration ofSaccharomyces brewing strains in response to heat and ethanol shock

SummaryThe effects of heat and ethanol shock on fatty acid composition and intracellular trehalose concentration of lager and ale brewing yeasts were examined. Exposure of cells to heat shock at 37°C or 10% (v/v) ethanol for 60 min resulted in a significant increase in the ratio of the total unsaturated to saturated fatty acyl residues and the intracellular trehalose concentration of cells. A similar increase in the amount of unsaturated fatty acids was observed in cells after 24 h of fermentation of 16°P (degree Plato) or 25°P wort, at which time more than 2% (v/v) ethanol was present in the growth medium. These results suggest that unsaturated fatty acids and high concentrations of intracellular trehalose may protect the cells from the inhibitory effects of heat and ethanol shock.

Effects of heat shock and ethanol stress on the viability of aSaccharomyces uvarum (carlsbergensis) brewing yeast strain during fermentation of high gravity wort

SummaryThe effects of heat shock and ethanol stress on the viability of a lager brewing yeast strain during fermentation of high gravity wort were studied. These stress effects resulted in reduced cell viability and inhibition of cell growth during fermentation. Cells were observed to be less tolerant to heat shock during the fermentation of 25°P (degree Plato) wort than cells fermenting 16°P wort. Degree Plato (oP) is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C. Relieving the stress effects of ethanol by washing the cells free of culture medium, improved their tolerance to heat shock. Cellular changes in yeast protein composition were observed after 24 h of fermentation at which time more than 2% (v/v) ethanol was present in the growth medium. The synthesis of these proteins was either induced by ethanol or was the result of the transition of cells from exponential phase to stationary phase of growth. No differences were observed in the protein composition of cells fermenting 16°P wort compared to those fermenting 25°P wort. Thus, the differences in the tolerance of these cells to heat shock may be due to the higher ethanol concentration produced in 25°P wort which enhanced their sensitivity to heat shock.

Transport kinetics of maltotriose in strains ofSaccharomyces

SummaryMaltotriose transport was studied in two brewers yeast strains, an ale strain 3001 and a lager strain 3021, using laboratory-synthesized14C-maltotriose. The maltotriose transport systems preferred a lower pH (pH 4.3) to a higher pH (pH 6.6). Two maltotriose transport affinity systems have been indentified. The high affinity system hasKm values of 1.3 mM for strain 3021 and 1.4 mM for strain 3001. The low affinity competitively inhibited by maltose and glucose withKi values of 58 mM and 177 mM. respectively, for strain 3021, and 55 mM and 147 mM, respectively, for strain 3001. Cells grown in maltotriose and maltose had higher maltotriose and maltose transport rates, and cells grown in glucose had lower maltortriose and maltose transport rates. Early-logarithmic phase cells transported glucose faster than either maltose or maltotriose. Cells harvested later in the growth phase had increased maltotriose and maltose transport activity. Neither strain exhibited significant differences with respect to maltose and maltotriose transport activity.

2-Deoxy-D-glucose resistant yeast with altered sugar transport activity.

The transport of glucose and maltose in Saccharomyces cerevisiae was observed to occur by both high and low affinity transport systems. A spontaneously isolated 2‐deoxy‐D‐glucose resistant mutant was observed to transport glucose and maltose only by the high affinity transport systems. Associated with this was an increase in the V max values, indicating derepression of the high affinity transport systems. The low affinity transport systems could not be detected. This mutant will be important in examining the repression regulatory and sugar transport mechanisms in yeast.

Biology of Ethanol-Producing Microorganisms

AbstractThe production of ethanol by microorganisms as a result of the fermentation of substrates such as sugars or starch is a process that predates recorded history. The uses of ethanol can be divided into a number of categories: (1) potable ethanol in beer, wine, sake, cider, and perry, a variety of fermented fruit juices, and in distilled beverages such as whiskey, gin, vodka, brandy, rum, and liquors; (2) solvent ethanol in the laboratory, in pharmaceutical preparations such as tonics and cough syrups, as a solvent for hop constituents, and in cosmetics; (3) as a cosurfactant in oil-water microemulsions; (4) as an antiseptic and sterilant; and (5) as a fuel in automobiles either en its own or more usually admixed with gasoline. It should not be forgotten that by far the largest volume of ethanol produced via fermentation is employed for potable purposes. Consequently, brewing, viticulture, and enology and distilled beverages are biotechnological industries that make a significant contribution to the ...