Cultivation of Saccharomyces cerevisiae in continuous culture

Summary Glucose limited growth of a respiratory deficient mutant of Saccharomyces cerevisiae was studied in continuous culture under steady state conditions. The maximal growth rate, the Michaelis constant, the cell yield, the maintenance coefficient and the ethanol yield of the growing cell population were determined. The steady state concentrations of cells, glucose and ethanol were measured as functions of the dilution rate and compared with theoretical predictions. A far-reaching agreement between theory and experiment was observed. The decrease of the cell yield in the range of low dilution rates is well explained by introducing the concept of maintenance energy in the general theory of continuous cultures. A deviation of the cell yield from the predicted values, which has been found in the range of high dilution rates, is discussed.     

Oscillatory metabolism of Saccharomyces cerevisiae in continuous culture

Short-period (40-50 min) synchronized metabolic oscillation was found in a continuous culture of yeast Saccharomyces cerevisiae under aerobic conditions at low-dilution rates. During oscillation, many parameters changed cyclically, such as dissolved oxygen concentration, respiration rate, ethanol and acetate concentrations in the culture, glycogen, ATP, NADH, pyruvate and acetate concentrations in the cells. These changes were considered to be associated with glycogen metabolism. When glycogen was degraded, the respiro-fermentative phase was observed, in which ethanol was produced and the respiration rate decreased. In this phase, the levels of intracellular pyruvate and acetate became minimum, ATP became high and intracellular pH at its lowest level. When glycogen metabolism changed from degradation to accumulation, the respiratory phase started, during which ethanol was re-assimilated from the culture and the respiration rate increased. Intracellular pyruvate and acetate became maximum, ATP decreased and the intracellular pH appeared high. These findings may indicate new aspects of the control mechanism of glycogen metabolism and how respiration and ethanol fermentation are regulated together under aerobic conditions.     

Mathematical analysis of a mechanism for autonomous metabolic oscillations in continuous culture of Saccharomyces cerevisiae.

Autonomous metabolic oscillations were observed in aerobic continuous culture of Saccharomyces cerevisiae. Experimental investigation of the underlying mechanism revealed that several pathways and regulatory couplings are involved. Here a hypothetical mechanism including the sulfate assimilation pathway, ethanol degradation and respiration is transformed into a mathematical model. Simulations confirm the ability of the model to produce limit cycle oscillations which reproduce most of the characteristic features of the system.     

Induction and elimination of oscillations in continuous cultures of Saccharomyces cerevisiae

Continuous cultures of Saccharomyces cerevisiae are known to exhibit oscillatory behavior in the oxidative region. Important findings of a series of experiments conducted to identify the causes for initiation of and the means for elimination of oscillations in these cultures are reported in this paper. These oscillations are seen to be connected to the growth kinetics of the microorganism and are induced at very low glucose concentrations and at dissolved oxygen (DO) levels that are neither high nor low (DO values between 20 and 78% air saturation at a dilution rate of 0.2 h(-1) and pH of 5.5 at 30 degrees C). The oscillatory behavior is encountered over a range of dilution rates (0.09-0.25 h(-1) at 30 degrees C for pH = 5.5 and DO = 50% air saturation). The oscillations can be eliminated by raising the DO level above a critical value or by lowering the DO level below a critical value.     

Influence of oxygen on the growth of Saccharomyces cerevisiae in continuous culture

Baker's yeast, Saccharomyces cerevisiae, was investigated for the combined influence of dissolved oxygen and glucose concentration in continuous culture. A reactor was operated at a range of dilution rates (0.1, 0.2, 0.25, 0.27, and 3.0 h(-1)), above and below the critical value that separates the oxidative and fermentation regions. For each dilution rate (D), steady states were established at each of five to ten different dissolved oxygen concentrations (DO) in the range of 0.01-5 mg/L. The use of on-line mass spectrometry facilitated the measurement of gaseous and dissolved O(2), CO(2), and ethanol. Intracellular carbohydrate, protein, RNA, DNA, lipid, and cytochrome concentrations were measured. Cell size measurements were reduced to specific surface areas. Cytochrome content showed up to 100% variation during a 20-day period of adaptation at D = 0.2 h(-1) to low DO. Eventually, the culture behaved the same at DO = 0.05 mg/L as it did initially at 3 mg/L. At D = 0.2, 0.25, and 0.27 h(-1), the transition between oxidation and fermentation was characterized by a critical DO which decreased with decreasing D. The X-D curves were shifted such that the critical D value was reduced with decreasing DO. Specific oxygen update rates varied with DO according to the saturation kinetics. Specific cell surface areas increased with decreasing DO. Cytochrome content generally decreased with decreasing DO, and Q(O(2) ) could be linearly related to the total cytochrome content, which exhibited a maximum at D = 0.27 h(-1).     

Effects of growth environment on recombinant plasmid stability in Saccharomyces cerevisiae grown in continuous culture

A recombinant strain of Saccharomyces cerevisiae, containing a 2-m-fragment-based plasmid (pYEa4) was grown under non-selective conditions in continuous culture. The decrease in the population carrying the plasmid-encoded auxotrophic marker, LEU2, was examined under different physiological conditions. The difference in growth rate (µ) between plasmid-free and plasmid-containing cells and the rate of plasmid segregation (R) were determined using a non-linear regression technique. Loss rates were greater in defined glucose-limited cultures than in complex glucose-limited cultures. Plasmid loss was µ-dominated in cultures grown on defined media whereas µ and R were co-dominant in cultures grown on complex medium. Loss rates increased with increasing dilution rate in complex glucose-limited cultures. The reverse was found in defined glucose-limited cultures. Plasmid retention and loss kinetics determined from defined magnesium-limited cultures were not significantly different from those observed in defined glucose-limited cultures. Although plasmid retention in defined phosphate-limited culture was not significantly different from that in defined glucose-limited culture, reduced R and increased µ indicated an alternative physiological effect of phosphate limitation on plasmid stability.     

Generation and maintenance of synchrony in Saccharomyces cerevisiae continuous culture

Cultures of Saccharomyces cerevisiae grown continuously produce an autonomous oscillation in many metabolic outputs. The most conveniently measured variable, i.e., dissolved oxygen concentration, oscillates with a period of 40-55 min. Previously we have identified two compounds capable of resetting phase, acetaldehyde and hydrogen sulfide. The phase-response curves constructed for acetaldehyde show a strong (Type 0) response at 3.0 mM and a weak (Type 1) response at 1.0 mM. Ammonium sulfide phase-response curves (pulse injected at 1.0 microM and 3.0 microM) revealed that sulfide is only an effective perturbation agent when endogenous sulfide concentrations are at a maximum. Also only Type 1 phase responses were observed. When the phase-response curve for sulfite (at 3.0 M) was constructed, phase responses were at a maximum at 60 degrees, indicating the possible involvement of sulfite in cell synchronization. It is concluded that endogenously produced acetaldehyde and sulfite tune the oscillation of mitochondrial energization state whereas sulfide mediates population synchrony.     

Effects of oxygen on invertase expression in continuous culture of recombinant Saccharomyces cerevisiae containing the SUC2 gene

The yeast SUC2 gene, cloned on a multicopy plasmid pRB58, was used to study the effect of oxygen on the invertase expression of the recombinant Saccharomyces cerevisiae. Glucose repression was not the only factor affecting the invertase expression. The results obtained from the single-stage continuous cultures under microaerobic conditions showed that invertase expression was also strongly dependent on oxygen availability, and moving from anaerobic to aerobic conditions led to a five-fold increase in specific invertase activity. However, the cell yields under anaerobic conditions were quite low compared to those under aerobic conditions. These opposite effects of oxygen on cell growth and gene expression offer a strategy for maximizing invertase productivity by a two-stage continuous culture. The first stage was operated at a low level of glucose, around 100 mg/l, under aerobic conditions in order to obtain a high yield of yeast biomass, and the second stage maintained anaerobic conditions with residual glucose levels of 50 mg/l to derepress and fully induce invertase expression. The two-stage continuous culture resulted in a 2.5-fold increase in invertase productivity over that of a single-stage continuous culture. Received: 28 July 1998 / Received revision: 22 September 1998 / Accepted: 7 November 1998     

Effects of culture conditions on ergosterol biosynthesis by Saccharomyces cerevisiae

Ergosterol is an essential component of yeast cells that maintains the integrity of the membrane. It was investigated as an important factor in the ethanol tolerance of yeast cells. We investigated the effects of brewing conditions on the ergosterol contents of S. cerevisiae K-9, sake yeast, several kinds of Saccharomyces cerevisiae that produce more than 20% ethanol, and X2180-1A, laboratory yeast. K-9 had a higher total ergosterol contents under all the conditions we examined than X2180-1A. Ethanol and hypoxia were found to have negative and synergistic effects on the total ergosterol contents of both strains, and significantly reduced the free ergosterol contents of X2180-1A but only slightly reduced those of K-9. The maintenance of free ergosterol contents under brewing conditions might be an important character of sake yeast strains. DNA microarray analysis also showed higher expression of ergosterol biosynthesis genes in K-9 than in X2180-1A.     

Lysine-overproducing mutants of Saccharomyces cerevisiae baker's yeast isolated in continuous culture.

Saccharomyces cerevisiae baker's yeast mutants which produce 3 to 17 times as much lysine as the wild type, depending on the nitrogen source, have been selected. The baker's yeast strain was growth in a pH-regulated chemostat in minimal medium with proline as the nitrogen source, supplemented with increasing concentrations of the toxic analog of the lysine S-2-aminoethyl-L-cysteine (AEC). The lysine-overproducing mutants, which were isolated as AEC-resistant mutants, were also resistant to high external concentrations of lysine and to alpha-aminoadipate and seemed to be affected in the lysine biosynthetic pathway but not in the biosynthetic pathways of other amino acids. Lysine overproduction by one of the mutants seemed to be due to, at least, the loss of repression of the homocitrate synthase encoded by the LYS20 gene. The mutant grew slower than the wild type, and its dough-raising capacity was reduced in in vitro assays, probably due to the toxic effects of lysine accumulation or of an intermediate produced in the pathway. This mutant can be added as a food supplement to enrich the nutritive qualities of bakery products, and its resistance to alpha-aminoadipate, AEC, and lysine can be used as a dominant marker.     

Oxygen-limited continuous culture and respiratory energy conservation in Escherichia coli.

Escherichia coli B was cultured continuously in succinate-minimal medium under conditions of oxygen limitation in the phauxostat. With decreasing oxygenation and consequent decreasing growth rates, the complement of terminal cytochrome oxidases changed as follows: high growth rates, cytochrome o; intermediate growth rates, cytochromes o and d; lowest growth rates, cytochromes o, d, and a1. Respiratory kinetics exhibited by nongrowing cell suspensions obtained from continuous cultures indicated that terminal oxidase activity was exhibited by cytochrome o (Km for O2 = 0.2 micron; Vmax = 1.1 to 1.5 mumol of O2 per nmol of cytochrome o per min) and cytochrome d (Km for O2 = 0.024 micron; Vmax = 0.7 mumol of O2 per nmol of cytochrome d per min). During oxygen-limited growth, the molar growth yield referred to respiration, and corrected for maintenance respiration [Yo(max)], was 12.6 g (dry weight) per g-atom of oxygen, not significantly different from the succinate-limited value of 12.0 g (dry weight) per g-atom of oxygen. The rate of maintenance respiration of the oxygen-limited culture was only 3.4 mg-atoms of O per g (dry weight) per h, some threefold less than that of the succinate-limited culture. Respiration-driven proton extrusion did not vary with the growth rate or with the complement of terminal oxidases (H+/O = 3.7; standard deviation, 0.07). We conclude that the content of terminal oxidases is without effect on the efficiency of respiratory energy conservation.     

Oscillatory behavior of Saccharomyces cerevisiae in continuous culture: I. Effects of pH and nitrogen levels

The appearance of sustained oscillations in bioreactor variables (biomass and nutrient concentrations) in continuous cultures of Saccharomyces cerevisiae indicates the complex nature of microbial systems, the inadequacy of current growth kinetic models, and the difficulties which may arise in bioprocess control and optimization. In this study we investigate continuous bioreactor behavior over a range of operating conditions (dilution rate, feed glucose concentration, feed ammonium concentration, dissolved oxygen, and pH) to determine the process requirements which lead to oscillatory behavior. We present new results which indicate that high feed ammonium concentrations may eliminate oscillations and that under oscillatory conditions ammonium levels are generally low and oscillatory as well. The effects of pH are complex and oscillations were only observed at pH values 5.5 and 6.5; no oscillations were observed at a pH of 4.5. Under our nominal operating conditions (feed glucose concentration 10 g/L, dilution rate 0.145 h(-1), feed ammonium concentration 0.0303M, dissolved oxygen level 50%, pH 5.5, and T = 30 degrees C) we found two possible final bioreactor states depending on the transient used to reach the nominal operating conditions. One of the states was oscillatory and characteristic of oxidative metabolism and the other was nonoscillatory and fermentative.     

The GTS1 gene product facilitates the self-organization of the energy metabolism oscillation in the continuous culture of the yeast Saccharomyces cerevisiae

To study the role of the GTS1 gene in the energy metabolism oscillation in continuous cultures of yeast from the physical aspect, time-series data of dissolved oxygen oscillations were analyzed by transforming them into power spectra and by creating two-dimensional trajectories using time delay embedding technique. We found that the wild-type cells organized themselves into a stable limit cycle oscillation and that the GTS1-deleted mutant, gts1Delta, usually showed transient oscillations whose power spectra resembled those of 1/f noise. Thus, we suggested that GTS1 plays an important role in the self-organization of the energy metabolism oscillation.     

Effects of sulphate-limited growth in continuous culture on the electron-transport chain and energy conservation in Escherichia coli K12.

Growth of Escherichia coli K12 in a chemostat was limited by sulphate concentrations lower than 300 muM. The synthesis of extracellular polysaccharide and a change in morphology accompanied sulphate-limited growth. Growth yields with respect to the amount of glycerol or oxygen consumed were sixfold and twofold lower respectively under these conditions than when growth was limited by glycerol. Sulphate-limited cells lacked the proton-translocating oxidoreduction segment of the electron-transport chain between NADH and the cytochromes, and particles prepared from these cells lacked the energy-dependent reduction of NAD+ by succinate, DL-alpha-glycerophosphate or D-lactate, suggesting the loss of site-I phosphorylation. Glycerol-limited cells contained cytochrome b556, b562 and o, ubiquinone and low concentrations of menaquinone. Sulphate limitation resulted in the additional synthesis of cytochromes d, a1, b558 and c550; the amount of ubiquinone was decreased and menaquinone was barely detectable. Non-haem iron and acid-labile sulphide concentrations were twofold lower in electron-transport particles prepared from sulphate-limited cells. Recovery of site-I phosphorylation could not be demonstrated after incubating sulphate-limited cells with or without glycerol, in either the absence or presence of added sulphate. The loss of site-I phosphorylation in sulphate-limited cells is discussed with reference to the accompanying alterations in cytochrome composition of such cells. Schemes are proposed for the functional organization of the respiratory chains of E. coli grown under conditions of glycerol or sulphate limitation.     

Estimating the maintenance energy and biomass concentration of Saccharomyces cerevisiae by continuous calorimetry

Continuous calorimetry has been applied to monitoring the heat evolution of Saccharomyces cerevisiae grown on d-glucose. The heat evolution, together with the energy and carbon balances, was used to evaluate the energetic efficiency of biomass, by-product biosynthesis, fermentative heat evolution as well as the maintenance energy of S. cerevisiae in ‘aerobic fermentation’ and ‘aerobic respiration’. In aerobic fermentation, under catabolite repression, the fraction of substrate energy converted to heat evolution, maintenance requirement, and biomass decreased with the increase of d-glucose concentration. The fraction of substrate energy converted to ethanol is the highest value and it could contribute up to 70% of the total substrate energy. In aerobic respiration, 43% of the total substrate energy was evolved as heat. While 50% of the total substrate energy was converted into biomass, only 7% of the total substrate energy was used for maintenance functions. The maintenance energy coefficient of S. cerevisiae was determined to be 0.427 MJ kg^?1 cell h^?1 (0.102 kcal g^?1 cell h^?1). For the first time, heat evolution together with yield-maintenance energy was used to predict biomass concentration during the fed-batch cultivation of S. cerevisiae.     

Effects of the cyanine dye 3,3''-dipropylthiocarbocyanine on mitochondrial energy conservation.

Mitochondrial respiration and oxidative phosphorylation were inhibited by the membrane potential probe 3,3'-dipropylthiocarbocyanine [diS-C3-(5)]. Evidence is presented that suggests that the dye acts as both an inhibitor of electron transport and an uncoupler of oxidative phosphorylation.