Morphological behaviour ofSaccharomyces cerevisiae during continuous fermentation

Summary Saccharomyces cerevisiae were observed to undergo drastic morphological changes when grown in continuous culture. Peak elongations and minimum cell volumes were found at intermediate dilution rates when it was believed the insitu glucose concentration was at its lowest. The shapes and sizes were reproducible and have been quantified at two different glucose feed concentrations.     

Culture fluorescence studies on aerobic continuous cultures ofSaccharomyces cerevisiae

Summary Fluorometric measurements were performed in continuous aerobic cultures ofSaccharomyces cerevisiae in order to study the effect of substrate concentration and residence time on the intracellular NADH-level. A modified Beyelermicrofluorometer probe (Beyeler et al. 1981) was used for the experiments. It was possible to use this sensor continuously up to five weeks without problems. The relative NADH-values obtained by the on-line monitoring of the NADH-dependent culture fluorescence were compared to the enzymatically determined NADH-content. Biomass estimation from fluorescence data was performed. During oxidative-reductive catabolism the deviation between calculated and measured data were below 5%. The differences between oxidative and oxidative-reductive catabolism were studied regarding glucose addition, dilution rate increase and aerobic-anaerobic transition. For synchronized continuous cultures, changes in dilution rate resulted in changes of the oscillating behaviour. Flow cytometric studies in comparison with fluorometric studies showed changes in budding behaviour during the oscillations.     

Microfluidic reactor for continuous cultivation of Saccharomyces cerevisiae

A diffusion-based microreactor system operated with a reaction volume of 8 μL is presented and characterized to intensify the process understanding in microscale cultivations. Its potential as screening tool for biological processes is evaluated. The advantage of the designed microbioreactor is the use for the continuous cultivation mode by integrating online measurement technique for dissolved oxygen (DO) and optical density (OD). A further advantage is the broaden application for biological systems. The bioreactor geometry was chosen to achieve homogeneous flow during continuous process operation. The device consisted of a microstructured top layer made of poly(dimethylsiloxane) (PDMS), which was designed and fabricated using UV-depth and soft lithography assembled with a glass bottom. CFD simulation data used for geometry design were verified via microparticle-image-velocimetry (μPIV). In the used microreactor geometry no concentration gradients occurred along the entire reaction volume because of rapid diffusive mixing, the homogeneous medium flow inside the growth chamber of the microreactor could be realized. Undesirable bubble formation before and during operation was reduced by using degassed medium as well as moistened and moderate incident air flow above the gas permeable PDMS membrane. Because of this a passive oxygen supply of the culture medium in the device is ensured by diffusion through the PDMS membrane. The oxygen supply itself was monitored online via integrated DO sensors based on a fluorescent dye complex. An adequate overall volumetric oxygen transfer coefficient K(L)a as well as mechanical stability of the device were accomplished for a membrane thickness of 300 μm. Experimental investigations considering measurements of OD (online) and several metabolite concentrations (offline) in a modified Verduyn medium. The used model organism Saccharomyces cerevisiae DSM 2155 tended to strong reactor wall growth resembling a biofilm.     

Potassium requirements ofSaccharomyces cerevisiae

The growth rate ofSaccharomyces cerevisiae was dependent on K⁺ content in culture medium in a certain range of K⁺ concentrations. Above the upper limit of the range, growth did not respond to K⁺ increase, and below the lower limit, yeast died. Rb⁺ and Na⁺ enhanced growth in the range of K⁺ dependence and decreased the K⁺ concentration below which cells died. Both Rb⁺ and Na⁺ became toxic above a certain Rb⁺/K⁺ and Na⁺/K⁺ cellular ratio.     

The chitin-glucan complex ofSaccharomyces cerevisiae

Differentiation of the cell wall ofSaccharomyces cerevisiae at the site of the future bud was followed. A lentil-like structure originates on the inner side of the cell wall during the first phase. At the same time, an electron-dense layer occurs at the boundary between the inner layer of the cell wall and the lentil-like structure. During the second phase granular material is accumulated at the lower side of the lentil-like structure. During the third phase the lentil-like structure is split apart due to proliferation of the granular material resulting in formation of the base of the encircling region. The marked electron-dense layer observed from the first phase is attached to the surface of the encircling region during differentiation of the latter. During the budding proper the outer layers of the cell wall protrude and the end of the encircling region, together with the adjacent electron-dense layer, acquire their definitive appearance of rings, observed as marked electron-transparent and electron-dense tears on ultrathin sections.     

Influence of oxygen tension on the physiology ofSaccharomyces cerevisiae in continuous culture

The effect of decreasing oxygen tensions on the physiology and composition ofSaccharomyces cerevisiae was studied in galactose-limited continuous cultures. Between oxygen tensions of 75 and 3 mm Hg the metabolism of galactose was oxidative and cell composition constant. Below 3 mm Hg a more fermentative metabolism was operative resulting in low cell yields and accumulation of ethanol in culture filtrates. This correlated with an increased alcohol dehydrogenase and a decreased particulate NADH oxidase activity of cell extracts. While the cellular contents of RNA and protein were not altered at low oxygen tensions there were marked changes in those of carbohydrates and lipids. Below 3 mm Hg there were decreases in most lipid constituents but those of membrane constituents such as sterol esters and phosphatides together with unsaturated fatty acids were most evident. ATP pool size was lowest and amino acid pool size highest at low oxygen tensions. The increased amino acid pool size was due to higher concentrations of arginine and aspartate.This work was supported by grant B/SR/5780 from the Science Research Council. We are grateful to the Brewers Society for a research studentship to B. Johnson. We thank Mr. A. Bradley and Mrs. C. Jones for competent technical assistance.     

The chitin-glucan complex ofSaccharomyces cerevisiae

Fractions of the cell wall ofSaccharomyces cerevisiae where a α-chtin-glucan composition was established, were examined under the electron microscope as well as with phase fluorescence. The results indicate that the α-chitin-glucan complex shows a fibrillar arrangement and is localized in the socalled encircling region of the bud scar which has a tear-like appearance and is electron-transparent in ultrathin sections. There are indications of the presence of chitin even in the primary septum.     

Batch cultivation ofSaccharomyces cerevisiae with increased content of Δ5’7 — Sterols

Changes of the physiological state were studied in a batch cultivation ofSaccharomyces cerevisiae, concentration of proteins and Δ5,7-sterols in yeast dry matter being taken as markers of these changes. Time variations of the markers were studied in relation to changing concentrations of carbon, nitrogen and phosphorus sources. A mathematical model of the batch cultivation was set up and identified. The method of parametric sensitivity was used to evaluate the effect of different technological parameters on the content of Δ5’7-sterols in yeast dry matter.     

The digestion ofSaccharomyces cerevisiae byAcanthamoeba castellanii

Summary The digestion ofSaccharomyces cerevisiae byAcanthamoeba castellanii, at different times after feeding, has been examined by cytochemical techniques at electron microscope level and by measurement of yeast viability. The measurement of viability, combined with cytochemistry is presented as a novel method of examining the progress of digestion. Particular attention has been given to the temporal development of digestion.Vacuoles, probably primary lysosomes, have been identified containing acid phosphatase activity within minutes of feeding and these accumulate around and fuse with phagocytic vacuoles. Acid phosphatase levels in the digestive vacuoles appeared highest at 20 to 40 minutes. Yeast digestion was observed and yeast viability began to decline at this time. Mixing of autophagic and heterophagic material was also observed. At least half of the yeast population was still viable after 90 minutes.Our method (p-nitrophenyl phosphate) of enzyme localization has demonstrated plasma membrane associated acid phosphatase activity.     

Temperature-sensitive flocculation during growth ofSaccharomyces cerevisiae

Summary Cell wall surface proteins were extracted from a temperature-sensitive flocculent strain ofSaccharomyces cerevisiae. Electrophoretic analysis identified two protein bands (28 and 43 kDa) present when grown at 21°C. These proteins were initially absent when the strain was grown at 37°C, but intensified after 6 days of growth concomitant with the onset of flocculation.     

Limited proteolysis ofSaccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Incubation ofSaccharomyces cerevisiae phosphoenolpyruvate carboxykinase with trypsin under native conditions cases a time-dependent loss of activity and the production of protein fragments. Cleavage sites determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and sequence analyses identified protease-sensitive peptide bonds between amino acid residues at positions 9–10 and 76–77. Additional fragmentation sites were also detected in a region approximately 70–80 amino acids before the carboxyl end of the protein. These results suggest that the enzyme is formed by a central compact domain comprising more than two thirds of the whole protein structure. From proteolysis experiments carried out in the presence of substrates, it could be inferred that CO₂ binding specifically protects position 76–77 from trypsin action. Intrinsic fluorescence measurements demonstrated that CO₂ binding induces a protein conformational change, and a dissociation constant for the enzyme CO₂ complex of 8.2±0.6 mM was determined     

Freeze-drying characteristics ofSaccharomyces cerevisiae andSaccharomyces carlsbergensis

The dispersal of yeast clumps to the unicellular state by certain sugars, does not increase the percentage survival after freeze-drying. Neither are those changes in cell-wall composition which occur upon ageing of the cell, and which are detectable by means of snail-gut enzymes, related to this cellular property. However, pre-treatment, with -mercaptoethanol, of a strain ofSaccharomyces carisbergensis increased the survival rate. This may be due to the reduction of certain sites in the cell wall. The oxygen consumption of yeast cultures before and after freeze-drying, agree with the hypothesis that low viabilities can arise from localized cellular damage which prevents cell reproduction by budding.     

Effect of Brestan on Saccharomyces cerevisiae during continuous cultivation

An increase in Brestan concentration in nutrient media decreased the content of protein, phosphorus, total ribonucleic acid, activity of pyruvate carboxylase and isocitrate lyase in cells ofSaccharomyces cerevisiae parent strain and respiratory deficient (RD) mutant while the trehalose content increased. The respiration quotient value for the RD mutant was higher than for the parent strain. The RD mutant lacked cytochromeaa ₃; cytochromec andb contents were lower than those of the parent strain.     

The application of dynamic calorimetry for monitoring growth ofSaccharomyces cerevisiae

Summary A dynamic calorimetric technique was investigated to determine the feasibility of monitoring cell growth by thermal measurements. Theoretical analysis of growth ofSaccharomyces cerevisiae on glucose showed that the correlation depends on cellular yield values but not on ethanol formation. Experiments withS. cerevisiae on a molasses-mineral salts medium resulted in a thermal yield of 4.4 kcal/g cells, consistent with our theoretical expectations.     

Electron microscopy of germinating ascospores ofSaccharomyces cerevisiae

The wall of mature ascospores ofSaccharomyces cerevisiae showed in sections under the electron microscope a dark outer layer and a lighter inner layer. The latter was composed of a greyish inner part and a light outer part. During germination, the spore grew out at one side and the dark outer layer was broken. Of the light inner layer, the inner greyish part became the wall of the vegetative cell, but the extented part of the cell had a new wall.     

Regulation of citrate synthase activity ofSaccharomyces cerevisiae

Citrate synthase activity ofSaccharomyces cerevisiae was determined by a radioactive assay procedure and the reaction product,¹⁴C-citric acid, was identified by chromatographic techniques. ATP, d-ATP, GTP and NADPH were most inhibitory to the citrate synthasein vitro. The activity was inhibited to a lesser extent by ADP, UTP, and NADP whereas, AMP and CTP were much less inhibitory. NADH, like NAD, glutamic acid, glutamine, arginine, ornithine, proline, aspartic acid and α-ketoglutarate exhibited no inhibition. These results have been discussed in the light of the role of citrate synthase for the energy metabolism and glutamic acid biosynthesis.     

Vacuolar (lysosomal) trehalase ofSaccharomyces cerevisiae

In the yeastSaccharomyces cerevisiae thePEP4 gene product, protease A, is responsible for activating all soluble vacuolar (lysosomal) enzymes. These vacuolar enzymes remain inactive inpep4 mutants. Vacuolar trehalase activity was diminished in such mutants as well. This suggests that the vacuolar (lysosomal) trehalase is processed in a manner similar to other vacuolar enzymes inS. cerevisiae.     

Transfer of isolated nuclei into protoplasts ofSaccharomyces cerevisiae

Cell nuclei were prepared from protoplasts of an adenine-requiring strain ofSaccharomyces cerevisiae, then purified in a discontinuous sucrose gradient, and applied to protoplasts of a recipient strain auxotrophic for uracil, leucine, and histidine. The transfer of the isolated nuclei into protoplasts was induced with polyethylene glycol. The main products of nuclear transfer in young complemented colonies were heterokaryons giving rise to parental type spontaneuos segregants on nutritionally complete medium. After several passages in minimal medium, however, the prototrophic colonies consisted exclusively of stable heterozygous diploid cells.     

Experimental bioenergetics ofSaccharomyces cerevisiae in respiration and fermentation

Aerobic growth of Saccharomyces cerevisiae on glucose was investigated, focusing on the heat evolution as it relates to biomass and ethanol synthesis. “Aerobic fermentation” and “aerobic respiration” were established respectively in the experimental system by performing batch and fed-batch experiments. “Balanced growth” batch cultivations were carried out with initial sugar concentrations ranging from 10 to 70 g/L, resulting in different degrees of catabolite repression. The fermentative heat generation was continuously monitored in addition to the key culture parameters such as ethanol production rate, CO₂ evolution rate, O₂ uptake rate, specific growth rate, and sugar consumption rate. The respective variations of the above quantities reflecting the variations in the catabolic activity of the culture were studied. This was done in order to evaluate the microbial regulatory system, the energetics of microbial growth including the rate of heat evolution and the distribution of organic substrate between respiration and fermentation. This study was supported by closing C, energy, and electron balances on the system. The comparison of the fractions of substrate energy evolved as heat (δ_h) with the fraction of available electrons transferred to oxygen (?_O2) indicated equal values of the two (0.46) in the aerobic respiration (fed-batch cultivation). However, the glucose effect in batch cultivations resulted in smaller ?_O2 than δ_h, while both values decreased in their absolute values. The evaluation of the heat energetic yield coefficients, together with the fraction of the available electrons transferred to O, contributed to the estimation of the extent of heat production through oxidative phosphorylation.