The structural complexity of the nitrogen source strongly affects both biomass and ethanol production by industrial strains of Saccharomyces cerevisiae, during fermentation in media containing glucose or maltose, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low glucose and maltose concentrations independent of nitrogen supplementation. At high sugar concentrations diauxie was not easily observed, and growth and ethanol production depended on the nature of the nitrogen source. This was different for baking and brewing ale and lager yeast strains. Sugar concentration had a strong effect on the shift from oxido-fermentative to oxidative metabolism. At low sugar concentrations, biomass production was similar under both peptone and casamino acid supplementation. Under casamino acid supplementation, the time for metabolic shift increased with the glucose concentration, together with a decrease in the biomass production. This drastic effect on glucose fermentation resulted in the extinction of the second growth phase, probably due to the loss of cell viability. Ammonium salts always induced poor yeast performance. In general, supplementation with a nitrogen source in the peptide form (peptone) was more positive for yeast metabolism, inducing higher biomass and ethanol production, and preserving yeast viability, in both glucose and maltose media, for baking and brewing ale and lager yeast strains. Determination of amino acid utilization showed that most free and peptide amino acids present, in peptone and casamino acids, were utilized by the yeast, suggesting that the results described in this work were not due to a nutritional status induced by nitrogen limitation.Key words: Amino acids, fermentation, nitrogen metabolism, peptides, Saccharomyces, yeast. -2863(9'8-32Yeast are able to use a wide variety of compounds as a carbon and nitrogen source 8,44,45 . Nitrogen is one of the main elements found in many macromolecules of living organisms, playing a central role in structure and function, and most organisms have elaborate control mechanisms to provide a constant supply of nitrogen 8,21,22,37,45 . Thus, a substantial proportion of cellular activity is concerned with procuring and assimilating nitrogen. It has been observed that ammonia, asparagine, glutamine and glutamate are preferentially used by yeast 21,37 . When these primary nitrogen sources are absent, or present in concentrations low enough to limit growth, other nitrogen sources such as nitrite, nitrate, amides, amino acids and peptides can be used. The utilization of secondary nitrogen sources requires the synthesis of specific-catabolic enzymes and permeases, the expression of which is highly regulated by a process known as nitrogen catabolite repression. The latter is prevented in the presence of a preferred nitrogen source 21,45 . Studies on the effect of nitrogen with only one compound as sole nitrogen and/or carbon source...

J. Inst. Brew. 112(2), 84-91, 2006Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish /stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.

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Biomass and ethanol production by industrial Saccharomyces cerevisiae strains were strongly affected by the structural complexity of the nitrogen source during fermentation in media containing galactose, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low galactose concentrations independent of nitrogen supplementation. At high sugar concentrations altered patterns of galactose utilisation were observed. Biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for baking and brewing ale and lager strains. Baking yeast showed improved galactose fermentation performance in the medium supplemented with casamino acids. High biomass production was observed with peptone and casamino acids for the ale brewing strain, however high ethanol production was observed only in the presence of casamino acids. Conversely, peptone was the nitrogen supplement that induced higher biomass and ethanol production for the lager brewing strain. Ammonium salts always induced poor yeast performance. The results with galactose differed from those obtained with glucose and maltose which indicated that supplementation with a nitrogen source in the peptide form (peptone) was more positive for yeast metabolism, suggesting that sugar catabolite repression has a central role in yeast performance in a medium containing nitrogen sources with differing levels of structural complexity.

Yeast adaptation to maltose utilization diminishes the repressing effect of glucose on maltose uptake. Furthermore, it affects both the rates and profiles of maltose, glucose and maltotriose uptake during high cell density brewer's wort fermentation. Yeast cells pre-grown in maltose, as sole carbon source, and harvested while the sugar is still present in the growth medium, are better adapted to utilize maltose. The adapted cells are less sensitive to glucose inhibition, and the uptake of glucose is inhibited in the early stages of fermentation. Cells grown for longer periods and harvested following maltose depletion, lose their ability to preferentially utilize maltose. In addition, they become more sensitive to glucose repression, and are able to utilize glucose faster than the cells harvested when maltose is still present in the medium. During the process of adaptation, maltose controls the induction of its own transport systems and appears to affect the biosynthesis of the glucose transport systems.

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In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (GGS1FDP1BYP1CIF1GLC6TSS1)‐encoded trehalose‐6‐phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50‐fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild‐type cells. However, it causes higher levels of glucose‐6‐phosphate, fructose‐6‐phosphate and also faster accumulation of fructose‐1,6‐bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and the wild‐type strain. Apparently, during the start‐up of fermentation hexokinase is more rate‐limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of d‐glucose and an equal concentration of radiolabelled l‐glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total d‐glucose measured (intracellular+periplasmic/extracellular) and the total l‐glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mm‐glucose to 0·5–2 mm in the wild‐type strain, ±10 mm in a hxk1Δ hxk2Δ glk1Δ and 2–3 mm in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1‐dependent control system is apparently able to restrict properly up to 50‐fold higher hexokinase activity. © 1998 John Wiley & Sons, Ltd.

The structural complexity of the nitrogen sources strongly affects biomass production and secretion of hydrolytic enzymes in filamentous fungi. Fusarium oxysporum and Aspergillus nidulans were grown in media containing glucose or starch, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids), peptides (peptone) and protein (gelatin). In glucose, when the initial pH was adjusted to 5.0, for both microorganisms, higher biomass production occurred upon supplementation with a nitrogen source in the peptide form (peptone and gelatin). With a close to neutrality pH, biomass accumulation was lower only in the presence of the ammonium salt. When grown in starch, biomass accumulation and secretion of hydrolytic enzymes (amylolytic and proteolytic) by Fusarium also depended on the nature of the nitrogen supplement and the pH. When the initial pH was adjusted to 5.0, higher growth and higher amylolytic activities were detected in the media supplemented with peptone, gelatin and casamino acids. However, at pH 7.0, higher biomass accumulation and higher amylolytic activities were observed upon supplementation with peptone or gelatin. Ammonium sulfate and casamino acids induced a lower production of biomass, and a different level of amylolytic enzyme secretion: high in ammonium sulfate and low in casamino acids. Secretion of proteolytic activity was always higher in the media supplemented with peptone and gelatin. Aspergillus, when grown in starch, was not as dependent as Fusarium on the nature of nitrogen source or the pH. The results described in this work indicate that the metabolism of fungi is regulated not only by pH, but also by the level of structural complexity of the nitrogen source in correlation to the carbon source.