Inge Russell

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.

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.

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.

Handbook of Brewing

Preface History of Industrial Brewing, Raymond G. Anderson Beer Styles: Their Origins and Classification, Charles Papazian An Overview of Brewing, Brian Eaton Water, David G. Taylor Barley and Malt, Geoffrey H. Palmer Adjuncts, Graham G. Stewart Hops, Trevor R. Roberts and Richard J.H. Wilson Yeast, Inge Russell Miscellaneous Ingredients in Aid of the Process, David S. Ryder and Joseph Power Brewhouse Technology, Kenneth A. Leiper and Michaela Miedl Brewing Process Control, Zane C. Barnes Fermentation, James H. Munroe Aging and Finishing, James H. Munroe Packaging: A Historical Perspective, Tom Fetters Packaging Technology, Alexander R. Dunn Microbiology and Microbiological Control in the Brewery, Fergus G. Priest Sanitation and Pest Control, Vernon E. Walter Brewery By-Products and Effluents, Nick J. Huige Beer Stability, Graham G. Stewart Quality, George Philliskirk Microbrewing, Johannes Braun and Brian H. Dishman Innovation and Novel Products, Inge Russell Index

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.

Changes in protein composition ofSaccharomyces brewing strains in response to heat shock and ethanol stress

SummaryHeat shock and ethanol stress of brewing yeast strains resulted in the induction of a set of proteins referred to as heat shock proteins (HSPs). At least six strongly induced HSPs were identified in a lager brewing strain and four HSPs in an ale brewing strain. Four of these HSPs with molecular masses of approximately 70, 38, 26 and 23 kDa were also identified in two laboratory strains ofSaccharomyces cerevisiae. The appearance of HSPs correlated with increased survival of strains at elevated temperatures and high concentrations of ethanol. These results suggest that HSPs may play a role in the ethanol and thermotolerance of yeasts. The properties of these proteins and membrane fatty acids in relation to heat and ethanol shock are being investigated.

Sugar uptake in a 2-deoxy-d-glucose resistant mutant ofSaccharomyces cerevisiae

SummaryThe non-metabolizable and toxic glucose analogue 2-deoxy-d-glucose (2-DOG) has been widely employed to screen for regulatory mutants which lack catabolite repression. A number of yeast mutants resistant to 2-DOG have recently been isolated in this laboratory. One such mutant, derived from aSaccharomyces cerevisiae haploid strain, was demonstrated to be derepressed for maltose, galactose and sucrose uptake. Furthermore, kinetic analysis of glucose transport suggested that the high affinity glucose transport system was also derepressed in the mutant strain. In addition, the mutant had an increased intracellular concentration of trehalose relative to the parental strain. These results indicate that the 2-DOG resistant mutant is defective in general glucose repression.

Regulation of glucose and maltose transport in strains ofSaccharomyces

SummaryGrowth of yeast cells on glucose resulted in complete inactivation of maltose transport and repression of the high affinity glucose transport system. When the cells were grown on maltose or subjected to substrate starvation, an increase in glucose and maltose transport was observed in both brewing and non-brewing yeast strains. The concentration of glucose employed in the growth medium was also observed to affect sugar transport activity. The higher the glucose concentration, the more pronounced the repressive effect. In addition, the time of growth of yeast on glucose or maltose also intermining the rate of sugar transport. These results are consistent with the repressive effect of glucose on the high affinity glucose and maltose transport systems.