The effects of glucose and oxygen on the cytochromes and metabolic activity of yeast batch cultures. I. Saccharomyces spp.

Saccharomyces cerevisiae and Saccharomyces carlsbergensis were grown in batch culture with and without oxygen control. The concentrations of A-, B- and C-type cytochromes of both yeasts were dependent on the oxygen concentration during growth as well as on the initial glucose concentration of the growth medium. S. cerevisiae cytochromes were maximal after growth in low glucose and low oxygen; S. carlsbergensis cytochromes were maximal after growth in low glucose and high oxygen. Except when glucose was in very low concentration, its catabolism by S. carlsbergensis was directed predominantly towards ethanolic fermentation regardless of the oxygen concentration. Growth rate, total cell mass and yield were maximal, and anabolism was closely balanced with catabolism, when glucose and oxygen of S. carlsbergensis cultures were both high. Under these conditions neither catabolism, respiratory or ethanolic, nor glucose uptake were maximal.     

The genusSaccharomyces (Meyen) Reess

Twenty-nine different strains of several fermentation type II species of the genusSaccharomyces, whose main common characteristic was complete fermentation of raffinose, were studied. These can be regarded as the most highly developed species of the genusSaccharomyces. Their common and differential morphological, physiological, biochemical and serological characteristics were studied. In addition to the usual criteria, factors which played a role in the history and formation of these species were taken into account. Large species were compared with typical strains chosen on the basis of statistical typing, e.g.Saccharomyces carlsbergensis Hansen, and small species were compared with type strains, e.g.Saccharomyces logos van Laer et Den. andSaccharomyces uvarum Beijer, as nomenelatural types. It was found that the whole group could be divided into two categories, one grouped roundSaccharomyces carlsbergensis Hansen and the other roundSaccharomyces uvarum Beijer., which overlapped at the place occupied bySaccharomyces logos van Laer et Den. Another group of intermediate strains isolated from grapes came betweenSaccharomyces logos andSaccharomyces uvarum. In future, closer attention will have to be paid to strains which do not give a serological reaction with serum against the typical speciesSaccharomyces carlsbergensis, i. e. to the speciesSaccharomyces monacensis Hansen andSaccharomyces mandshuricus Saito.     

Visualization of chromosome-like structures in protoplasts of the yeast

Structures resembling chromosomes have been observed in protoplasts of Saccharomyces carlsbergensis during mitotic and meiotic division. The stage of visualization is prolonged in meiosis, and bivalents can be counted. The haploid chromosome number of S. carlsbergensis is between 22–26.     

Invertase biosynthesis by Saccharomyces carlsbergensis in batch and continuous cultures

The biosynthesis of invertase by Saccharomyces carlsbergensis LAM 1068 was studied in relation to its glucose effect at both unsteady and steady states of growth. Experimental correlations between the dilution rate and invertase specific activity (E/X) in chemostat, cultures led to an optimum for the enzyme synthesis at a particular intermediate growth rate. The value of E/X increased from 1.1 (U/mg biomass) in batch cultures to 13 (U/mg biomass) in chemostat cultures. A mutant strain A3 showed the highest value for E/X = 25 (U/mg biomass) at high dilution rates where glucose repression was observed with the wild strain.     

The action of visible radiation on the formation and properties of Saccharomyces ascospores

Summary Light in the blue and green bands of the spectrum inhibited ascospore formation in both Saccharomyces cerevisiae and S. carlsbergensis. Ascospores which were produced failed to stain with malachite green and mature spores formed in the dark lost their staining ability when exposed to light in these bands. It is thought that this is due to an alteration brought about in the molecular organization of the spore wall, so that the damaged walls become permeable to molecules of considerably greater size. Red light was without effect on S. cerevisiae, but stimulated sporulation in S. carlsbergensis. This response seemed to be strictly under the control of the yeast cell, and of a totally different nature from the injurious character of shorter wave lengths.     

Accumulation of Nicotinamide Adenine Dinucleotide in Baker’s Yeast by Secondary Culture

A study was made to determine a method for the production of NAD using baker’s yeast, and a suitable secondary culture condition for the accumulation of NAD was established. From the study the following results were obtained: Nicotinamide, nicotinic acid and adenine were effective on the accumulation of NAD. However, ribose or tryptophan — one of the precursor of NAD — was not effective. NaF, KCN or NaN₃ — metabolic inhibitors — inhibited the accumulation of NAD. Baker’s yeast obtained from commercial source was cultured secondarily in the medium containing 0.3% adenine, 0.6% nicotinamide in 0.2 M K₂HPO₄ (50% fresh yeast was added), pH 4.5. Under this optimal condition, NAD content reached about 12 mg/g dry cells (corresponding to 2.0 mg/ml medium), and it corresponded to about 20 times that of the initial content.     

The genusSaccharomyces (Meyen) Reess

The authors submit a taxonomic evaluation of an intermediate group of strains between the speciesSaccharomyces carlsbergensis Hansen andSaccharomyces cerevisiae Hansen. The material consisted of atypical strains of “bottom” brewer’s yeasts and the synonymous strainsSaccharomyces monacensis Hansen andSaccharomyces mandshuricus Saito. It was found that there were two different serological types in the speciesSaccharomyces carlsbergensis, one of which was characterized by the presence of antigen “C” and was typical for this species, while the other possessed antigen “M” and was grouped roundSaccharomyces monacensis. This second serological type merges with a group of strains which gives only one third fermentation of raffinose, so that it is actually an intermediate betweenSaccharomyces cerevisiae Hansen andSaccharomyces carlsbergensis Hansen and indicates the course of progressive development from the former species to the latter. No close similarity was found betweenSaccharomyces mandshuricus Saito and some of the strains of the transitional group or typical representatives of the two main species, and the authors therefore consider that there is some obscurity as to its synonymity withSaccharomyces carlsbergensis.     

Participation of vacuoles in regulation of levels of K+, Mg2+ and orthophosphate ions in cytoplasm of the yeast Saccharomyces carlsbergensis

Intracellular distributions of K⁺, Mg²⁺ and orthophosphate under various conditions of cultivation or incubation of the yeast Saccharomyces carlsbergensis were studied by differential extraction of ion pools. The decisive role of vacuolar compartmentation of ions in regulation of K⁺, Mg²⁺ and orthophosphate levels in the yeast cytoplasm was shown. The content of intracellular K⁺ and Mg²⁺ in yeast increased or decreased primarily depending on the increase or decrease in the vacuolar ion pool. The levels of K⁺ and Mg²⁺ in the cytoplasm were practically unchanged. Vacuoles were involved in regulation of Mn²⁺ concentration in the cytoplasm of the yeast S. carlsbergensis accumulating this ion in the presence of glucose. Alongside the vacuolar compartmentation, the chemical compartmentation, i. e. formation of bound Mg²⁺, Mn²⁺ and K⁺ was, evidently, also involved in the control of ion levels in the cytoplasm. The orthophosphate level in the yeast cytoplasm was regulated by its accumulation in vacuoles and biosynthesis of inorganic polyphosphates in these organelles. The biosynthesis of low-molecular weight polyphosphates occurred parallel to the accumulation of Mg²⁺ or Mn²⁺ in vacuoles, thus confirming the availability of the other mechanism for the transport of these ions through the tonoplast differing from the transport mechanism through the plasmalemma.     

Production of phenylacetylcarbinol by various yeast species

Measurements of tolerance to the addition of benzaldehyde were carried out with six yeast species:Hansenula anomala, Brettanomyces vini Peynaud et Domercq, strain X,Saccharomyces carlsbergensis, Saccharomyces cerevisiae R XII,Saccharomyces ellipsoideus andTorula utilis. The techniques used were: comparison of evolution of carbon dioxide with and without benzaldehyde and production of phenylacetylcarbinol after a single addition of benzaldehyde (0.2%) and after four additions (total of 0.8%). The highest decarboxylase activity in the presence of benzaldehyde was found withHansenula anomala, Saccharomyces carlsbergensis andSaccharomyces cerevisiae. InHansenula anomala, benzaldehyde caused a 16% inhibition of fermentation, in the other cultures inhibition lay between 35.5 and 63.2%. After a single addition of benzaldehyde the greatest amount of phenylacetylcarbinol was formed byHansenula anomala, Saccharomyces carlsbergensis andSaccharomyces cerevisiae. The yield of phenylacetylcarbinol in these cases was between 46.0% and 51.5 weight% (calculated on added benzaldehyde). During fementation with a higher benzaldehyde concentration,Saccharomyces carlsbergensis utilized 70% aldehyde for the formation of phenylacetylcarbinol while the rest was mostly reduced to benzylalcohol. Phenylacetylcarbinol was estimated after extraction of the fermentation medium with ether polarographically and polarimetrically, benzaldehyde was estimated polarographically directly in the medium.     

Effects of Fusariotoxin T-2 on Saccharomyces cerevisiae and Saccharomyces carlsbergensis

A Fusarium metabolite, T-2 toxin, inhibits the growth of Saccharomyces carlsbergensis and Saccharomyces cerevisiae. The growth inhibitory concentrations of T-2 toxin were 40 and 100 μg/ml, respectively, for exponentially growing cultures of the two yeasts. S. carlsbergensis was more sensitive to the toxin and exhibited a biphasic dose-response curve. Addition of the toxin at 10 μg/ml of S. carlsbergensis culture resulted in a retardation of growth as measured turbidimetrically, after only 30 to 40 min. This action was reversible upon washing the cells free of the toxin. The sensitivity of the yeasts to the toxin was dependent upon the types and concentrations of carbohydrates used in the growth media. The sensitivity of the cells to the toxin decreased in glucose-repressed cultures. These results suggest that T-2 toxin interferes with mitochondrial functions of these yeasts.     

Characterization of a regulatory mutant of fructose 1,6-bisphosphatase in Saccharomyces carlsbergensis.

Summary The fdp mutation has been localized on the genome of Saccharomyces carlsbergensis, on chromosome II, between lys2 and tyr1, at a map distance of 31 centimorgan from lys2.Since the fdp mutant does not grow on glucose, fructose, mannose and sucrose, hexose transport and a number of enzymes of carbon metabolism were tested, but no significant differences could be found between the wild type and the mutant. Only the regulatory properties of glycogen synthetase are changed in the mutant, but it is doubtfull whether this can explain its phenotype.The disorganization of carbon metabolism of the mutant upon addition of glucose to the medium was analyzed in more detail. The most prominent feature observed until now is the accumulation of free glucose and hexose phosphates in the cell. This result indicates that somehow the feedback control between hexose transport and metabolism is impaired. Hexose phosphates are known to be toxic to many cells, including yeast. Therefore, accumulation of hexose phosphates in the presence of glucose in the medium, can explain the absence of growth on this carbon source.     

Stoffwechsel-Regulation aerober Zellsuspensionen vonSaccharomyces carlsbergensis nach Glucosezugabe

Zusammenfassung Wird eine ausgehungerte aerobe Zellsuspension vonSaccharomyces carlsbergensis mit Glucose gefüttert, dann ist eine deutliche Stimulierung des O₂-Verbrauchs zu registrieren. Mit Hilfe verschiedener Meßmethoden wurde der Verlauf dieser nicht-linearen O₂-Abnahme analysiert und mit den gleichzeitig auftretenden Konzentrationsänderungen einiger glykolytischer Metabolite korreliert.Dabei zeigt sich, daß ca. 20–60 Sekunden nach Glucosezugabe die Rate des O₂-Verbrauchs abnimmt und gleichzeitig eine deutliche Äthanol-Synthese einsetzt, obgleich der O₂-Partialdruck zu diesem Zeitpunkt noch etwa halbmaximal ist.

---

Metabolite-regulation of aerobic cell-suspension ofSaccharomyces carlsbergensis after feeding with glucose. I. Shift from respiration to aerobic fermentation

Summary After feeding with glucose, an aerobic starved cell-suspension ofSaccharomyces carlsbergensis shows an increasing of oxygen consumption. This stimulation is not linear during the transition from high to low oxygen-level.For this aerobic phase the regulated fluxes of some metabolites are analyzed. It could be shown that with highest oxygen consumption an ethanol synthesis is starting.

---

---

Herrn Prof. Dr.A. Betz danke ich an dieser Stelle für viele wertvolle Hinweise zur Interpretation der hier beschriebenen Ergebnisse. Die gewissenhafte technische Assistenz erfolgte durch FrauR. Hinrichs. Die Durchführung dieser Arbeit wurde durch Sachbeihilfen der Stiftung Volkswagenwerk und der Deutschen Forschungsgemeinschaft ermöglicht.     

Dual control of invertase Biosynthesis in chemostat culture

In a previous study on the chemostat culture of Saccharomyces carlsbergensis, maximum invertase specific activity was observed at an intermediate dilution rate. A possible regulation mechanism, assuming there are simultaneous effects of induction and repression on two sites of the operator loci for invertase formation, is proposed which might account for the observed curve of the dilution rate effect.     

Production of phenylacetylcarbinol in various yeast species

Summary Production of phenylacetylcarbinol (PAC) was measured in various yeast species. The yeast strains tested were cultivated under submerged conditions in a medium containing corn steep and sucrose as the main components; sucrose, acetaldehyde and benzaldehyde were added to the grown cultures. In a first series of experiments the initial rate of PAC production, i.e. the PAC production determined 30 min after the addition of benzaldehyde was determined in 38 yeast strains, mostly of the generaSaccharomyces andCandida. The amount of PAC produced varied from zero (12 strains) to 1.24 mg ml^–1. In a second series of experiments, 15 strains, which in the first series had shown a higher PAC production, were further tested. Sucrose, acetaldehyde and benzaldehyde were added to the cultures until the PAC production ceased. The highest PAC production (6.3 mg ml^–1) was reached in the strainSaccharomyces carlsbergensis Budvar; the production was slightly lower in 4 strains of the generaSaccharomyces, Candida andHansenula.     

Stoffwechselregulation in Durchfluß-systemen

Zusammenfassung Am Beispiel der unter kontinuierlicher Substratzufuhr erfolgenden Äthanol-Synthese in Hefe (Saccharomyces carlsbergensis) konnte gezeigt werden, daß unabhängig von der exogenen Substratmenge der Speicherstoffwechsel einer linearen Regelfunktion unterliegt. Das Verhältnis zwischen Äthanolbildung aus exogenem und endogenem Material ist derart ausbalanciert, daß die Gesamtbilanz erhalten bleibt, nach der 50–60% der zugeführten Glucosemenge im Äthanol wiederzufinden sind.

---

Metabolic control in flow systemsII. Regulation of ethanol-synthesis and pool turnover in yeast under continuous substrate infusion

Summary The synthesis of ethanol under continuous infusion of d-glucose to anaerobic Saccharomyces carlsbergensis demonstrates very distinctly that turnover of the carbohydrate pool has the characteristics of a linear regulation, independent of the exogenous substrate amounts. The relation between glycolysis and pool turnover is balanced exctly so that 50–60% of glucose infused will be synthesized to ethanol.

     

Effects of thiamine and pyridoxine on respiratory activity in Saccharomyces carlsbergensis strain 4228

Studies were made to elucidate the relationship among the thiamine-induced growth inhibition, decrease in cellular vitamin B₆ content and respiratory deficiency in Saccharomyces carlsbergensis strain 4228 [Nakamura et al., Biochem. Biophys. Res. Commun. 59, 771–776 (1974)]. Addition of pyridoxine to the thiamine-added culture at the beginning or in the course of cultivation brought about appearance of cytochrome spectra and the increase in the activity of heme-containing enzymes and in respiratory activity (Q _{O 2}). The effects of pyridoxine occurred prior to the restoration of growth. Pyridoxine was effective even in the presence of high levels of glucose in the growth medium (not less than 3%). On the basis of these results, the mechanism of the effects of thiamine and pyridoxine was discussed.     

Sterol-binding polysaccharides of Rhizopus arrhizus, Penicillium roquefortii and Saccharomyces carlsbergensis

Polysaccharides that bind with sterols and render them water-soluble were isolated from two mycelial fungi, Rhizopus arrhizus and Penicillium roquefortii and a yeast Saccharomyces carlsbergensis. The polysaccharides from R. arrhizus and S. carlsbergensis were accompanied by small quantities of phosphorus, protein and lipid, none of which significantly influenced the binding of sterol to polysaccharide. The chemical composition and sterol-binding properties of the polysaccharides from the filamentous species were almost identical, but differed significantly from those of the yeast polysaccharide. The principal sterol-binding polysaccharide of S. carlsbergensis was identified as a mannan and that of the filamentous fungi as a glucan(s). The binding capacity of the purified yeast polysaccharide was almost two-fold greater than that of R. arrhizus and P. roquefortii.     

Influence of glucose concentration on the physiology and lipid composition of some yeasts

The fatty acid contents of the “Crabtree-positive” yeastsSaccharomyces cerevisiae, S. carlsbergensis andS. delbrueckii decreased with increasing concentrations of glucose in the medium. These lower values were due to a lower content of sterol esters and phosphatides inS. cerevisiae, and of sterol esters inS. carlsbergensis. In contrast the fatty acid contents of the Crabtree-negativeS. fragilis, Schwanniomyces occidentalis andCandida utilis increased with increasing concentrations of glucose and inCandida utilis this was due almost entirely to a higher content of triglycerides. This work was supported in part by grant B/SR/5780 from the Science Research Council. We are grateful to the Brewer's Society for a Research Scholarship to Mr. B. Johnson. We thank Mr. A. Bradley for competent technical assistance.     

Kinetics of Δ5,7-sterol accumulation during growth ofSaccharomyces cerevisiae

Saccharomyces cerevisiae accumulates Δ^5,7-sterols up to 4 mg per g biomass. The differential rate of sterol synthesis continually increases during growth, its value only being decreased at sterol levels higher than 30 mg per g biomass. The specific rate of sterol synthesis reaches a broad maximum during the growth phase. The gradual sterol accumulation pattern is dominant in cultures growing both on fermentable and nonfermentable carbon sources and is modulated by glucose repression. Limited feeding with sucrose has a significantly greater negative impact on sterol accumulation than feeding with ethanol as a carbon source.     

A novel nicotinamide adenine dinucleotide control strategy for increasing the cell density of Haemophilus parasuis

Haemophilus parasuis is the causative agent of Glässer's disease and is a major source of economic losses in the swine industry each year. To enhance the production of an inactivated vaccine against H. parasuis, the availability of nicotinamide adenine dinucleotide (NAD) must be carefully controlled to ensure a sufficiently high cell density of H. parasuis. In the present study, the real-time viable cell density of H. parasuis was calculated based on the capacitance of the culture. By assessing the relationship between capacitance and viable cell density/NAD concentration, the NAD supply rate could be adjusted in real time to maintain the NAD concentration at a set value based on the linear relationship between capacitance and NAD consumption. The linear relationship between cell density and addition of NAD indicated that 7.138 × 10⁹ NAD molecules were required to satisfy per cell growth. Five types of NAD supply strategy were used to maintain different NAD concentration for H. parasuis cultivation, and the results revealed that the highest viable cell density (8.57, OD₆₀₀) and cell count (1.57 × 10¹⁰ CFU/mL) were obtained with strategy III (NAD concentration maintained at 30 mg/L), which were 1.46- and 1.45- times more, respectively, than cultures with using NAD supply strategy I (NAD concentration maintained at 10 mg/L). An extremely high cell density of H. parasuis was achieved using this NAD supply strategy, and the results demonstrated a convenient and reliable method for determining the real-time viable cell density relative to NAD concentration. Moreover, this method provides a theoretical foundation and an efficient approach for high cell density cultivation of other auxotroph bacteria.