Oxygen Requirements of the Food Spoilage Yeast Zygosaccharomyces bailii in Synthetic and Complex Media

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.     

Aerobic sugar metabolism in the spoilage yeast Zygosaccharomyces bailii

Despite the importance of some Zygosaccharomyces species as agents causing spoilage of food, the carbon and energy metabolism of most of them is yet largely unknown. This is the case with Zygosaccharomyces bailii. In this study the occurrence of the Crabtree effect in the petite-negative yeast Z. bailii ATCC 36947 was investigated. In this yeast the aerobic ethanol production is strictly dependent on the carbon source utilised. In glucose-limited continuous cultures a very low level of ethanol was produced. In fructose-limited continuous cultures ethanol was produced at a higher level and its production increased with the dilution rate. As a consequence, on fructose the onset of respiro-fermentative metabolism caused a reduction in biomass yield. An immediate aerobic alcoholic fermentation in Z. bailii was observed during the transition from sugar limitation to sugar excess, both on glucose and on fructose. The analysis of some key enzymes of the fermentative metabolism showed a high level of acetyl-CoA synthetase in Z. bailii growing on fructose. At high dilution rates, the activities of glucose- and fructose-phosphorylating enzymes, as well as of pyruvate decarboxylase and alcohol dehydrogenase, were higher in cells during growth on fructose than on glucose.     

Oxygen requirements of yeasts

Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly mu max, 0.03 and 0.05 h-1, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth.     

Zygosaccharomyces lentus: a significant new osmophilic, preservative-resistant spoilage yeast, capable of growth at low temperature

Zygosaccharomyces lentus is a yeast species recently identified from its physiology and 18S ribosomal sequencing (Steels et al. 1999).The physiological characteristics of five strains of this new yeast so far isolated were investigated, particularly those of technical significance for a spoilage yeast, namely temperature range, pH range, osmotolerance, sugar fermentation, resistance to food preservatives such as sorbic acid, benzoic acid and dimethyldicarbonate (DMDC; Velcorin). Adaptation to benzoic acid, and growth in shaking and static culture were also investigated. Zygosaccharomyces lentus strains grew over a wide range of temperature (4-25 degrees C) and pH 2.2-7.0. Growth at 4 degrees C was significant. Zygosaccharomyces lentus strains grew at 25-26 degrees C in static culture but were unable to grow in aerobic culture close to their temperature maximum. All Z. lentus strains grew in 60% w/v sugar and consequently, are osmotolerant. Zygosaccharomyces lentus strains could utilize sucrose, glucose or fructose as a source of fermentable sugar, but not galactose. Zygosaccharomyces lentus strains were resistant to food preservatives, growing in sorbic acid up to 400 mg l-1 and benzoic acid to 900 mg l-1 at pH 4.0. Adaptation to higher preservative concentrations was demonstrated with benzoic acid. Resistance to DMDC was shown to be greater than that of Z. bailii and Saccharomyces cerevisiae. This study confirms that Z. lentus is an important food spoilage organism potentially capable of growth in a wide range of food products, particularly low pH, high sugar foods and drinks. It is likely to be more significant than Z. bailii in the spoilage of chilled products.     

Oxygen requirements of yeasts.

Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly mu max, 0.03 and 0.05 h-1, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth.     

Resistance of some wine spoilage yeasts to combinations of ethanol and acids present in wine

The most important factors affecting microbial growth in an alcoholic beverage are the ethanol content and the low pH. The effectiveness of different organic acids in conjunction with ethanol concentration in controlling growth of yeasts was determined for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Brettanomyces lambicus, Pichia anomala, Zygosaccharomyces bailii, Saccharomycodes ludwigii and Kluyveromyces thermotolerans in malt extract broth (MEB). The results are summarized as undissociated concentrations of different acids required to inhibit growth of yeasts in MEB containing 10% (v/v) ethanol. About half the amount of undissociated malic or tartaric acid is necessary for inhibition of the yeasts, compared with acetic and lactic acid but the concentrations of acid necessary to inhibit growth were generally very high and unrealistic in wines for controlling growth of most of the yeasts tested. All the yeasts tested were able to grow in acidified non- or low alcoholic beverages but, at higher ethanol concentrations, Sacch. cerevisiae and Zygosacch. bailii have the greatest spoilage potential.     

Reactions of Saccharomyces cerevisiae and Zygosaccharomyces bailii to sulphite

Sulphite inhibited growth of all four yeasts studied, Zygosaccharomyces bailii NCYC 563 being most sensitive and Saccharomyces cerevisiae NCYC 431 the least. Vertical Woolf-Eadie plots were obtained for initial velocities of 35S accumulation by all four yeasts suspended in high concentrations of sulphite. Equilibrium levels of 35S accumulation were reached somewhat faster with strains of S. cerevisiae than with those of Z. bailii. With all four yeasts, the greater the extent of 35S accumulation, the larger was the decline in internal pH value. Growth of S. cerevisiae TC8 and Z. bailii NCYC 563, but to a lesser extent of S. cerevisiae NCYC 431 and Z. bailii NCYC 1427, was inhibited when mid exponential-phase cultures were supplemented with 1.0 or 2.0 mM-sulphite, the decrease in growth being accompanied by a decline in ethanol production. Unless growth was completely inhibited, the sulphite-induced decline in growth was accompanied by production of acetaldehyde and additional glycerol.     

Sour rot-damaged grapes are sources of wine spoilage yeasts

Yeast species of sound and sour rot-damaged grapes were analysed during fermentation and grape ripening in the vineyard, using general and selective culture media. During 2003 and 2004 vintages, microvinifications were carried out with sound grapes to which different amounts of grapes with sour rot were added. The wine spoilage species Zygosaccharomyces bailii was only recovered during fermentations with sour rot, reaching 5.00 log CFU mL(-1) (2003) and 2.48 log CFU mL(-1) (2004) at the end of fermentation. The study of yeast populations during the sour rot ripening process (2005 vintage) showed that the veraison-damaged grapes always exhibited higher total yeast counts and a much greater diversity of species. From a total of 22 ascomycetous species, 17 were present only in damaged grapes. The most frequent species were Issatchenkia occidentalis and Zygoascus hellenicus. The spoilage species Z. bailii and Zygosaccharomyces bisporus were consistently isolated exclusively from damaged grapes. This work demonstrates that one of the most dangerous wine spoilage species, Z. bailii, is strongly associated with sour rot grapes and survives during fermentation with Saccharomyces cerevisiae. The use of selective media provides a more accurate characterization of grape contamination species.     

High-cell-density cultivation of yeasts on disaccharides in oxygen-limited batch cultures

Many facultatively fermentative yeast species exhibit a "Kluyver effect": even under oxygen-limited growth conditions, certain disaccharides that support aerobic, respiratory growth are not fermented, even though the component monosaccharides are good fermentation substrates. This article investigates the applicability of this phenomenon for high-cell-density cultivation of yeasts. In glucose-grown batch cultures of Candida utilis CBS 621, the onset of oxygen limitation led to alcoholic fermentation and, consequently, a decrease of the biomass yield on sugar. In maltose-grown cultures, alcoholic fermentation did not occur and oxygen-limited growth resulted in high biomass concentrations (90 g dry weight L(-1) from 200 g L(-1) maltose monohydrate in a simple batch fermentation). It was subsequently investigated whether this principle could also be applied to Kluyveromyces species exhibiting a Kluyver effect for lactose. In oxygen-limited, glucose-grown chemostat cultures of K. wickerhamii CBS 2745, high ethanol concentrations and low biomass yields were observed. Conversely, ethanol was absent and biomass yields on sugar were high in oxygen-limited chemostat cultures grown on lactose. Batch cultures of K. wickerhamii grown on lactose exhibited the same growth characteristics as the maltose-grown C. utilis cultures: absence of ethanol formation and high biomass yields. Within the species K. marxianus, the occurrence of a Kluyver effect for lactose is known to be strain dependent. Thus, K. marxianus CBS 7894 could be grown to high biomass densities in lactose-grown batch cultures, whereas strain CBS 5795 produced ethanol after the onset of oxygen limitation and, consequently, yielded low amounts of biomass. Because the use of yeast strains exhibiting a Kluyver effect obviates the need for controlled substrate-feeding strategies to avoid oxygen limitation, such strains should be excellently suited for the production of biomass and growth-related products from low-cost disaccharide-containing feedstocks. (c) 1996 John Wiley & Sons, Inc.     

Glucose respiration and fermentation in Zygosaccharomyces bailii and Saccharomyces cerevisiae express different sensitivity patterns to ethanol and acetic acid

In the yeast Zygosaccharomyces bailii ISA 1307, respiration and fermentation ofglucose were exponentially inhibited by ethanol, both processes displaying similar sensitivity tothe alcohol. Moreover, the degree of inhibition on fermentation was of the same magnitude as thatreported for Saccharomyces cerevisiae. Acetic acid also inhibited these two metabolicprocesses in Z. bailii , with the kinetics of inhibition again being exponential. However,inhibition of fermentation was much less pronounced than in S. cerevisiae. The valuesestimated with Z. bailii for the minimum inhibitory concentration of acetic acid rangedfrom 100 to 240 mmol l⁻¹ total acetic acid compared with values of near zeroreported for S. cerevisiae. The inhibitory effects of acetic acid on Z. bailii were notsignificantly potentiated by ethanol.     

Variation in yeast mitochondrial activity associated with asci

An increase in mitochondrial membrane potential (DeltaPsim) and mitochondrially produced 3-hydroxy (3-OH) oxylipins was experienced in asci of the nonfermentative yeasts Galactomyces reessii and Lipomyces starkeyi and the fermentative yeasts Pichia farinosa and Schizosaccharomyces octosporus. Strikingly, asci of Zygosaccharomyces bailii showed no increase in mitochondrial activity (DeltaPsim and oxylipin production). As expected, oxygen deprivation only inhibited ascus formation in those yeasts with increased ascus mitochondrial activity. We conclude that ascus formation in yeasts is not always dependent on mitochondrial activity. In this case, fermentation may provide enough energy for ascus formation in Z. bailii.     

Kinetics of growth and sugar consumption in yeasts

An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts.Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called Crabtree effect probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect inS. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast.S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions.Non-Saccharomyces yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeastCandida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.     

关于四个酵母菌种对乙醇耐受性的研究

本文报道了从存活,生长,发酵能力和氢离子渗漏等角度研究和比较四种酵母菌(Zygosaccharomyces bailii NCYC 1427,Saccharomyces cerevisiae NCYC 431,Harisenula anomala NCYC 711和 Kloeckera apiculata NCYC 468)对乙醇的耐受性。乙醇对酵母在上述特性的抑制程度,取决于乙醇的浓度。Z．Bailii NCYC 1427是四个酵母中乙醇耐受性最强的。在2M的乙醇溶液中,其存活率与无乙醇的对照组一样高。在低于1.5M的乙醇中,其生长率高于 S．Cerevisiae NCYC 431,氢离子渗入细胞的速率增长则低于 S．Cerevisiae 和 H．Anomala。S．Cerevisiae NCYC 431对于乙醇抑制生长的效应耐受性很强,在低于IM的乙醇溶液中,其发酵能力下降得较为缓慢,存活率几乎没有变化。H．Anomala NCYC 711在生长、发酵能力和氢离子渗漏方面对乙醇的抑制作用均表现敏感,但其存活率变化却与S．Cerevisiae NCYC 431很相似。K．Apiculata NCYC 468在存活、生长和发酵能力方面对乙醇的效应是四个菌种中最敏感的。研究结果还表明,乙醇对生长、发酵能力和氢离子渗漏的作用比它对存活的影响大得多。环境条件也影响酵母对乙醇的耐受性。     

Yeast community structures and dynamics in healthy and Botrytis-affected grape must fermentations

Indigenous yeast population dynamics during the fermentation of healthy and Botrytis-affected grape juice samples from two regions in Greece, Attica and Arcadia, were surveyed. Species diversity was evaluated by using restriction fragment length polymorphism and sequence analyses of the 5.8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of cultivable yeasts. Community-level profiles were also obtained by direct analysis of fermenting samples through denaturing gradient gel electrophoresis of 26S rDNA amplicons. Both approaches revealed structural divergences in yeast communities between samples of different sanitary states or geographical origins. In all cases, Botrytis infection severely perturbed the bioprocess of fermentation by dramatically altering species heterogeneity and succession during the time course. At the beginning and middle of fermentations, Botrytis-affected samples possessed higher levels of biodiversity than their healthy counterparts, being enriched with fermentative and/or spoilage species, such as Zygosaccharomyces bailii and Issatchenkia spp. or Kluyveromyces dobzhanskii and Kazachstania sp. populations that have not been reported before for wine fermentations. Importantly, Botrytis-affected samples exposed discrete final species dominance. Selection was not species specific, and two different populations, i.e., Saccharomyces cerevisiae in samples from Arcadia and Z. bailii in samples from Attica, could be recovered at the end of Botrytis-affected fermentations. The governing of wine fermentations by Z. bailii is reported for the first time and could elucidate the origins and role of this particular spoilage microbe for the wine industry. This is the first survey to compare healthy and Botrytis-affected spontaneous fermentations by using both culture-based and -independent molecular methods in an attempt to further illuminate the complex yeast ecology of grape must fermentations.     

Homofermentative lactate production cannot sustain anaerobic growth of engineered Saccharomyces cerevisiae: possible consequence of energy-dependent lactate export

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.     

Effect of oxygen on morphogenesis and polypeptide expression by Mucor racemosus.

The morphology of Mucor racemosus in cultures continuously sparged with nitrogen gas was investigated. When appropriate precautions were taken to prevent oxygen from entering the cultures, the morphology of the cells was uniformly yeastlike irrespective of the N2 flow rate. When small amounts of oxygen entered the cultures the resulting microaerobic conditions evoked mycelial development. Polypeptides synthesized by aerobic mycelia, microaerobic mycelia, anaerobic yeasts, and yeasts grown in a CO2 atmosphere were compared by two-dimensional gel electrophoresis. The results indicated that a large number of differences in polypeptide expression exist when microaerobic mycelia or anaerobic yeasts are compared with aerobic mycelia and that these alterations correlate with a change from an oxidative to a fermentative metabolic mode. Relatively few differences in polypeptide composition exist when microaerobic cells are compared with anaerobic cells, but these changes correlate with a change from the mycelial to the yeast morphology. We hypothesize that oxygen regulates the expression of polypeptides involved in both the metabolic mode and in morphogenesis.     

Reduction of volatile acidity of wines by selected yeast strains

Herein, we isolate and characterize wine yeasts with the ability to reduce volatile acidity of wines using a refermentation process, which consists in mixing the acidic wine with freshly crushed grapes or musts or, alternatively, in the incubation with the residual marc. From a set of 135 yeast isolates, four strains revealed the ability to use glucose and acetic acid simultaneously. Three of them were identified as Saccharomyces cerevisiae and one as Lachancea thermotolerans. Among nine commercial S. cerevisiae strains, strains S26, S29, and S30 display similar glucose and acetic acid initial simultaneous consumption pattern and were assessed in refermentation assays. In a medium containing an acidic wine with high glucose-low ethanol concentrations, under low oxygen availability, strain S29 is the most efficient one, whereas L. thermotolerans 44C is able to decrease significantly acetic acid similar to the control strain Zygosaccharomyces bailii ISA 1307 but only under aerobic conditions. Conversely, for low glucose-high ethanol concentrations, under aerobic conditions, S26 is the most efficient acid-degrading strain, while under limited-aerobic conditions, all the S. cerevisiae strains studied display acetic acid degradation efficiencies identical to Z. bailii. Moreover, S26 strain also reveals capacity to decrease volatile acidity of wines. Together, the S. cerevisiae strains characterized herein appear promising for the oenological removal of volatile acidity of acidic wines.     

Steady-state and transient-state analyses of aerobic fermentation in Saccharomyces kluyveri

Some yeasts, such as Saccharomyces cerevisiae, produce ethanol at fully aerobic conditions, whereas other yeasts, such as Kluyveromyces lactis, do not. In this study we investigated the occurrence of aerobic alcoholic fermentation in the petite-negative yeast Saccharomyces kluyveri that is only distantly related to S. cerevisiae. In aerobic glucose-limited continuous cultures of S. kluyveri, two growth regimens were observed: at dilution rates below 0.5 h(-1) the metabolism was purely respiratory, and at dilution rates above 0.5 h(-1) the metabolism was respiro-fermentative. The dilution rate at which the switch in metabolism occurred, i.e. the critical dilution rate, was 66% higher than the typical critical dilution rate of S. cerevisiae. The maximum specific oxygen consumption rate around the critical dilution rate was found to 13.6 mmol (g dry weight)(-1) h(-1) and the capacity of the pyruvate dehydrogenase-bypass pathway was estimated to be high from in vitro enzyme activities; especially the specific activity of acetyl-CoA synthetase was much higher than in S. cerevisiae at all tested conditions. Addition of glucose to respiring cells of S. kluyveri led to ethanol formation after a delay of 20-50 min (depending on culture conditions prior to the pulse), which is in contrast to S. cerevisiae that ferments immediately after glucose addition.     

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621

The role of pyruvate metabolism in the triggering of aerobic, alcoholic fermentation in Saccharomyces cerevisiae has been studied. Since Candida utilis does not exhibit a Crabtree effect. this yeast was used as a reference organism. The localization, activity and kinetic properties of pyruvate carboxylase (EC 6.4.1.1), the pyruvate dehydrogenase complex and pyruvate decarboxylase (EC 4.1.1.1) in cells of glucose-limited chemostat cultures of the two yeasts were compared. In contrast to the general situation in fungi, plants and animals, pyruvate carboxylase was found to be a cytosolic enzyme in both yeasts. This implies that for anabolic processes, transport of C4-dicarboxylic acids into the mitochondria is required. Isolated mitochondria from both yeasts exhibited the same kinetics with respect to oxidation of malate. Also, the affinity of isolated mitochondria for pyruvate oxidation and the in situ activity of the pyruvate dehydrogenase complex was similar in both types of mitochondria. The activity of the cytosolic enzyme pyruvate decarboxylase in S. cerevisiae from glucose-limited chemostat cultures was 8-fold that in C. utilis. The enzyme was purified from both organisms, and its kinetic properties were determined. Pyruvate decarboxylase of both yeasts was competitively inhibited by inorganic phosphate. The enzyme of S. cerevisiae was more sensitive to this inhibitor than the enzyme of C. utilis. The in vivo role of phosphate inhibition of pyruvate decarboxylase upon transition of cells from glucose limitation to glucose excess and the associated triggering of alcoholic fermentation was investigated with 31P-NMR. In both yeasts this transition resulted in a rapid drop of the cytosolic inorganic phosphate concentration. It is concluded that the relief from phosphate inhibition does stimulate alcoholic fermentation, but it is not a prerequisite for pyruvate decarboxylase to become active in vivo. Rather, a high glycolytic flux and a high level of this enzyme are decisive for the occurrence of alcoholic fermentation after transfer of cells from glucose limitation to glucose excess.     

Causes of conductance change in yeast cultures.

The conductance change due to growth of Saccharomyces cerevisiae Y112, Zygosaccharomyces bailii M and Rhodotorula rubra NCYC 63 in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source was compared with that in a medium containing L-asparagine as sole nitrogen source. Decreases in conductance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with L-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for conductance change in yeast cultures is the uptake of charged ammonium ions as nitrogen source and the reaction of protons with pH buffer compounds. Rhodotorula rubra cultures with L-asparagine as sole carbon source caused large increases in conductance with growth. Chemical analyses of culture filtrates showed that this increase in conductance was due to use of L-asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic needs as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in conductance.