A biochemically structured model for Saccharomyces cerevisiae.

A biochemically structured model for the aerobic growth of Saccharomyces cerevisiae on glucose and ethanol is presented. The model focuses on the pyruvate and acetaldehyde branch points where overflow metabolism occurs when the growth changes from oxidative to oxido-reductive. The model is designed to describe the onset of aerobic alcoholic fermentation during steady-state as well as under dynamical conditions, by triggering an increase in the glycolytic flux using a key signalling component which is assumed to be closely related to acetaldehyde. An investigation of the modelled process dynamics in a continuous cultivation revealed multiple steady states in a region of dilution rates around the transition between oxidative and oxido-reductive growth. A bifurcation analysis using the two external variables, the dilution rate, D, and the inlet concentration of glucose, S(f), as parameters, showed that a fold bifurcation occurs close to the critical dilution rate resulting in multiple steady-states. The region of dilution rates within which multiple steady states may occur depends strongly on the substrate feed concentration. Consequently a single steady state may prevail at low feed concentrations, whereas multiple steady states may occur over a relatively wide range of dilution rates at higher feed concentrations.     

Dynamic effects related to steady-state multiplicity in continuous Saccharomyces cerevisiae cultivations

The behavioral differences between chemostat and productostat cultivation of aerobic glucose-limited Saccharomyces cerevisiae were investigated. Three types of experiments were conducted: a chemostat, where the dilution rate was shifted up or down in stepwise manner; and a productostat, with either stepwise changed or a rampwise increased ethanol setpoint, i.e., an accelero-productostat. The transient responses from chemostat and productostat experiments were interpreted using a simple metabolic flux model. In a productostat it was possible to obtain oxido-reductive steady states at dilution rates far below Dcrit due to a strong repression of the respiratory system. However, these steady states could not be obtained in a chemostat, since a dilution rate shift-down from an oxido-reductive steady state led to a derepression of the respiratory system. It can therefore be concluded that the range of dilution rates where steady-state multiplicity can be obtained differs depending on the operation mode and that this dilution rate multiplicity range may appear larger in a productostat than in a chemostat. A more narrow multiplicity range, however, was obtained when the productostat was operated as an accelero-productostat.     

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.     

Modeling the growth and proteinase A production in continuous cultures of recombinant Saccharomyces cerevisiae

Overexpression of the homologous protein proteinase A (PrA) in Saccharomyces cerevisiae has been achieved by inserting the PrA gene (PEP4) with its own promoter on a 2mu multicopy plasmid. With this system the specific PrA production rate was found to be described well by a linear function of the oxidative glucose metabolism, the reductive glucose metabolism, and the oxidative ethanol metabolism, with a significant lower yield resulting from the reductive glucose metabolism compared with the oxidative glucose metabolism. To describe the experimental data, a simple mathematical model has been set up. The model is based on an assumption of a limited respiratory capacity as suggested by Sonnleitner and K?ppeli but extended to describe production of an extracellular protein. The model predicts correctly the critical dilution rate to be between 0.15 and 0.16 h(-1), the decrease in the biomass yield above the critical dilution rate, and the production of proteinase A at different dilution rates. Both the experimental data and model simulations suggest that the optimum operating conditions for protein production is just at the critical dilution rate. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 447-454, 1997.     

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).     

初始条件对高浓度乙醇连续发酵过程的影响及振荡行为提高发酵效率的机理分析

前期实验在稀释速率为0.027h-1的高浓度乙醇连续发酵过程中,发现了一种长周期、宽振幅的参数振荡现象。本实验进一步考察了不同稀释速率下的连续发酵过程,发现在稀释速率为0.04h-1条件下,也能出现类似的振荡现象;在稀释速率为0.027h-1或0.04h-1的条件下,改变系统的初始状态可以得到振荡和稳态两种不同的发酵过程。比较振荡和稳态过程的实验数据后,发现在稀释速率为0.04h-1的条件下,与稳态过程相比,振荡过程的平均残糖浓度降低了14.8%,平均乙醇浓度提高了12.6%,平均设备生产强度提高了12.3%。进一步分析表明:与稳态过程相比,振荡过程动力学行为不仅存在滞后,而且在相同残糖和乙醇浓度条件下,所对应的平均比生长速率提高了53.8%。     

Chemostat-cultivated Escherichia coli at high dilution rate: multiple steady states and drift

The representation of metabolic network reaction kinetics in a scaled, polynomial form can allow for the prediction of multiple steady states. The polynomial formalism is used to study chemostat-cultured Escherichia coli which has been observed to exhibit two multiple steady states under ammonium ion-limited growth conditions: a high cell density-low ammonium ion concentration steady state and a low cell density-high ammonium ion concentration steady state. Additionally, the low-cell-density steady state has been observed to drift to the high-cell-density steady state. Inspection of the steady-state rate expressions for the ammonium ion transport/assimilation network (in polynomial form) suggests that at low ammonium ion concentrations, two steady states are possible. One corresponds to heavy use of the glutamine synthetase-glutamate synthase (GLNS-GS) branch and the second to heavy use of the glutamate dehydrogenase (GDH) branch. Realization of the predicted intracellular steady states is also found to be dependent on the parameters of the transport process. Moreover, the two steady states differ in where their energy intensity lies. To explain the drift, GLNS, which is inducible under low ammonium ion concentrations, is suggested to be a "memory element." A chemostat-based model is developed to illustrate that perturbations in dilution rate can lead to drift between the two steady states provided that the disturbance in dilution rate is sufficiently large and/or long in duration.     

Effects of a hexokinase II deletion on the dynamics of glycolysis in continuous cultures of Saccharomyces cerevisiae

In glucose-limited aerobic chemostat cultures of a wild-type Saccharomyces cerevisiae and a derived hxk2 null strain, metabolic fluxes were identical. However, the concentrations of intracellular metabolites, especially fructose 1,6-bisphosphate, and hexose-phosphorylating activities differed. Interestingly, the hxk2 null strain showed a higher maximal growth rate and higher Crabtree threshold dilution rate, revealing a higher oxidative capacity for this strain. After a pulse of glucose, aerobic glucose-limited cultures of wild-type S. cerevisiae displayed an overshoot in the intracellular concentrations of glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-bisphosphate before a new steady state was established, in contrast to the hxk2 null strain which reached a new steady state without overshoot of these metabolites. At low dilution rates the overshoot of intracellular metabolites in the wild-type strain coincided with the immediate production of ethanol after the glucose pulse. In contrast, in the hxk2 null strain the production of ethanol started gradually. However, in spite of the initial differences in ethanol production and dynamic behaviour of the intracellular metabolites, the steady-state fluxes after transition from glucose limitation to glucose excess were not significantly different in the wild-type strain and the hxk2 null strain at any dilution rate.     

Cultivation of Saccharomyces cerevisiae in continuous culture

Summary Growth of Saccharomyces cerevisiae was investigated under aerobic conditions in a glucose limited chemostat. The steady state concentrations of cells, glucose and ethanol were measured in dependence of the dilution rate. The growth rate showed a biphasic dependence from the glucose concentration. A shift from respiratory to fermentative metabolism (Crabtree-effect) altering heavily the cell yield and the ethanol yield took place in the range of dilution rates between 0.3 h^-1 and 0.5 h^-1. Therefore the classical theory of continuous cultures is not applicable on aerobic growth of Saccharomyces cerevisiae under glucose limitation without introducing further premises. On the other hand the steady state cell concentration as a function of the dilution rate fits well the theoretically calculated curves, if cells are cultivated under conditions where only fermentation or respiration is possible.     

Actual and potential rates of substrate oxidation and product formation in continuous cultures ofChromatium vinosum

Kinetics of electron-donor oxidation, storage-polymer formation and growth were studied in continuous cultures ofChromatium under conditions of balanced growth as well as during transient states.Under steady-state conditions, glycogen was accumulated at all dilution rates. This observation is consistent with previously postulated ideas about an ineffective glycogen-synthesis regulation.Upon perturbing the steady states, brought about by injection of extra sulfide into steady-state cultures, the following phenomena were observed immediately, irrespective of the dilution rate: the specific rate of sulfide oxidation increased to the value found in batch cultures, the sulfur-oxidation rate was decreased, the specific glycogen-synthesis rate increased, the increment being higher the lower the dilution rate, but an increase in the specific growth rate, if any, was below the limit of detection. The inverse relationship between the specific rates of glycogen synthesis and growth after removing the substrate limitation is to be explained by a shortage of intermediates, rather than by a growth-rate dependent intrinsic glycogen-synthesis limitation, because upon complete inhibition of growth a further increase in the rate of glycogen synthesis was observed. Essayed in this way, identical glycogen-synthesis rates were found at all dilution rates.Competitive advantages of such an apparently not adapted metabolism in environments with diurnal fluctuations in substrate concentrations are discussed.Non-Standard Abbreviations N_c cell nitrogen - TS total sugar - PHB poly--hydroxybutyrate - D dilution rate - S_R reservoir concentration of the growth-limiting substrate - CAP chloramphenicol     

Influence of dilution rate and induction of cloned gene expression in continuous fermentations of recombinant yeast

The effects of growth rate on cloned gene product synthesis in recombinant Saccharomyces cerevisiae have been studied in continuous culture. The plasmid employed contains a yeast GAL10-CYC1 hybrid promoter directing expression of the E. coli lacZ gene. beta-Galactosidase production was therefore controlled by the yeast galactose regulatory circuit, and the induction process and its effects were studied at the various dilution rates. At all dilution rates plasmid stability decreased with induction of lacZ gene expression. In some instances, two induced "steady states" were observed, the first 10-15 residence times after induction and the second after 40-50 residence times. The second induced steady state was characterized by greater biomass concentration and lower beta-galactosidase specific activity relative to the first induced "steady-state." beta-Galactosidase specific activity and biomass concentration increased as dilution rate was reduced, and despite lower flow rate and plasmid stability, overall productivity (activity/L/hr) was substantially higher at low dilution rate. Important factors influencing all of the trends were the glucose and galactose (inducer) concentrations in the vessel and inducer metabolism.     

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.     

Observed quasi-steady kinetics of yeast cell growth and ethanol formation under very high gravity fermentation condition

Using a generalSaccharomyces cerevisiae as a model strain, continuous ethanol fermentation was carried out in a stirred tank bioreactor with a working volume of 1,500 mL. Three different gravity media containing glucose of 120, 200 and 280 g/L, respectively, supplemented with 5 g/L yeast extract and 3 g/L peptone, were fed into the fermentor at different dilution rates. Although complete steady states developed for low gravity medium containing 120 g/L glucose, quasi-steady states and oscillations of the fermented parameters, including residual glucose, ethanol and biomass were observed when high gravity medium containing 200 g/L glucose and very high gravity medium containing 280 g/L glucose were fed at the designated dilution rate of 0.027 h⁻¹. The observed quasi-steady states that incorporated these steady states, quasi-steady states and oscillations were proposed as these oscillations were of relatively short periods of time and their averages fluctuated up and down almost symmetrically. The continuous kinetic models that combined both the substrate and product inhibitions were developed and correlated for these observed quasi-steady states.     

The effect of vitamins and amino acids on glucose uptake in aerobic chemostat cultures of three Saccharomyces cerevisiae strains

In the respiro-fermentative region of aerobic chemostat cultures at steady state, Saccharomyces cerevisiae CBS 8066 produced high concentrations of ethanol with concomitant low levels of residual glucose which followed Monod kinetics. By contrast, very high residual glucose concentrations were observed in cultures of S. cerevisiae strains ATCC 4126 and NRRL Y132 at dilution rates above 60% of the washout dilution rate, resulting in much lower ethanol concentrations, even though clearly glucose-limited at lower dilution rates in the respiratory region. The addition of a vitamin mixture resulted in decreased residual glucose concentrations in respiro-fermentative cultures of all three strains, but the effect was much more pronounced with strains ATCC 4126 and NRRL Y132. Meso-inositol was mainly responsible for this effect, although with strain ATCC 4126 other vitamins as well as an amino acid mixture were also required to minimise the steady-state residual glucose levels. The residual glucose concentration in continuous culture was, therefore, greatly dependent on the growth factor requirements of the particular yeast strain, which apparently increased on increasing the dilution rate into the respiro-fermentative region. The strain differences with respect to growth factor requirements at high dilution rates, which were not evident at low dilution rates, had a profound effect on the kinetics of glucose assimilation in aerobic chemostat culture.     

Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state and transient continuous cultures

The production of the extracellular alkaline protease Savinase (EC 3.4.21.62) and glucose uptake in a non-sporulating strain of Bacillus clausii were investigated by analysing steady-state and transients during continuous cultivations. The specific production rate was found to have an optimum at a dilution rate between 0.14 and 0.17 h(-1), whereas the yield of Savinase on glucose was found to increase with decreasing specific growth rate. A linear relationship between the ribosomal RNA content and the specific production rate was found, indicating that the translational capacity may be limiting for product formation. The dynamics of the production of Savinase were studied during step changes in the dilution rate. During a step down in the dilution rate the specific production rate decreased immediately until it reached a new steady value. During a step-up an initial cease in the production rate was observed, but when glucose stopped to accumulate the production rate was regained. The glucose uptake was further investigated when chemostat cultures growing at different dilution rates were exposed to glucose pulses. The maximal glucose uptake capacity was found to be dependent on the initial specific growth rate. Furthermore, the adaptation to high glucose concentrations was faster at high dilution rates than at low dilution rates.     

自絮凝酵母SPSC01在组合反应器系统中酒精连续发酵的研究

建立了一套由四级磁力搅拌发酵罐串联组成、总有效容积4000mL的小型组合生物反应器系统 ,其中一级罐作为种子培养罐。以脱胚脱皮玉米粉双酶法制备的糖化液为种子培养基和发酵底物 ,进行了自絮凝颗粒酵母酒精连续发酵的研究。种子罐培养基还原糖浓度为100g L ,添加 (NH₄)₂HPO₄ 和KH₂PO₄ 各 20g L ,以0.017h^-1 的恒定稀释速率流加 ,并溢流至后续酒精发酵系统。发酵底物初始还原糖浓度 220g/L ,添加 (NH₄)₂HPO₄ 15g/L和KH₂PO₄2 5g/L ,流加至第一级发酵罐 ,稀释速率分别为 0.017、0.025、0.033、0.040和0.05 0h^-1。实验数据表明 ,自絮凝颗粒酵母在各发酵罐中呈部分固定化状态 ,在稀释速率0.040h^-1 条件下 ,发酵系统呈一定的振荡行为 ,其他四个稀释速率实验组均能够达拟稳态。当稀释速率不超过 0 0 33h^-1 ,流出末级发酵罐的发酵液中酒精浓度可以达到 12 % (V/V)以上 ,残还原糖和残总糖分别在 0 11%和 0 35 % h^-1,流出末级发酵罐的发酵液中酒精浓度可以达到12%（V/V）以上,残还原糖和残总糖分别在0.11%和0.35%（W/V）以下。在稀释速率为0.033h^-1时,计算发酵系统酒精的设备生产强度指标为3.32（g·L^-1·h^-1）,与游离酵母细胞传统酒精发酵工艺相比,增加约1倍。     

Mechanism of the stimulatory effect of fructose on ethanol oxidation in perfused rat liver.

The stimulatory effect of fructose on ethanol oxidation was studied in livers from fasted rats perfused with Krebs-Henseleit-bicarbonate buffer in a non-recirculating system. Two series of experiments were performed: (A) ethanol was infused with stepwise increasing concentrations (0.1-20 mM) in the presence of 4 mM fructose; (B) fructose was infused with stepwise increasing concentrations (0.5-10 mM) in the presence of 2 mM ethanol. From measured metabolic rates the following parameters were calculated: energy-rich phosphates consumed for fructose metabolism which were provided from oxidative phosphorylation (delta approximately P); reducing equivalents derived from stimulated ethanol utilization which were disposed by mitochondrial oxidation (delta2H). Under the various conditions studied a linear relationship between these parameters was observed. The ratio delta approximately P/delta2H was about 2.0. It is suggested that fructose stimulates ethanol oxidation indirectly by increasing the energy consumption of the liver due to the production of glucose from fructose. Consequetnly, the rate of oxidative phosphorylation is increased and, therefore, the capacity of the respiratory chain for oxidizing reducing equivalents derived from ethanol is enhanced. The data support the more general hypothesis that the rate of ethanol oxidation depend upon the rate of hepatic energy consumption in a given metabolic state.     

Effect of Specific Growth Rate on Fermentative Capacity of Baker’s Yeast

The specific growth rate is a key control parameter in the industrial production of baker’s yeast. Nevertheless, quantitative data describing its effect on fermentative capacity are not available from the literature. In this study, the effect of the specific growth rate on the physiology and fermentative capacity of an industrial Saccharomyces cerevisiae strain in aerobic, glucose-limited chemostat cultures was investigated. At specific growth rates (dilution rates, D) below 0.28 h⁻¹, glucose metabolism was fully respiratory. Above this dilution rate, respirofermentative metabolism set in, with ethanol production rates of up to 14 mmol of ethanol · g of biomass⁻¹ · h⁻¹ at D = 0.40 h⁻¹. A substantial fermentative capacity (assayed offline as ethanol production rate under anaerobic conditions) was found in cultures in which no ethanol was detectable (D < 0.28 h⁻¹). This fermentative capacity increased with increasing dilution rates, from 10.0 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.025 h⁻¹ to 20.5 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.28 h⁻¹. At even higher dilution rates, the fermentative capacity showed only a small further increase, up to 22.0 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.40 h⁻¹. The activities of all glycolytic enzymes, pyruvate decarboxylase, and alcohol dehydrogenase were determined in cell extracts. Only the in vitro activities of pyruvate decarboxylase and phosphofructokinase showed a clear positive correlation with fermentative capacity. These enzymes are interesting targets for overexpression in attempts to improve the fermentative capacity of aerobic cultures grown at low specific growth rates.     

Continuous culture of Thiorhodaceae

Summary In sulfide limited continuous culture of a marine isolate of Chromatium vinosum, sulfide was undetectable in steady states below dilution rates of 0.06h^-1, that is 1/2 of the maximum specific growth rate. In the same range, sulfur is assumed to attain the role of the growth rate limiting substrate. Furthermore, it could be shown that the rate of sulfur oxidation is a function of the surface area of the sulfur globules rather than of the sulfur concentration. In completely filled chemostats, steady states were obtainable only at dilution rates not exceeding 0.09 h^-1. In the presence of a nitrogen flushed gas phase, steady states were obtained at dilution rates approaching the maximum specific growth rate (0.12h^-1). This phenomenon is ascribed to the particular sulfide tolerance of our strain of Chromatium vinosum. The saturation constant and the inhibition constant (lowest, respectively highest total sulfide concentration at which the specific growth rate is equal to one-half of the maximum specific growth rate in the absence of inhibition) were 0.007 mM and 0.85 mM, respectively.The ecological significance of the data is discussed.Contribution No. 2406 from the Woods Hole Oceanographic Institution.