Uptake and accumulation of B-group vitamers in Saccharomyces cerevisiae in ethanol-stat fed-batch culture

The uptake and accumulation of the B-group vitamins thiamine, riboflavin, nicotinamide, pantothenic acid and pyridoxine in Saccharomyces cerevisiae was studied by gradually increasing the specific dosage of vitamins in an ethanol-stat fed-batch culture. Thiamine, nicotinamide, pantothenic acid, and pyridoxine were almost completely taken up at low vitamin dosages. Thiamine was determined to be the major accumulating form of vitamin B₁ while most of the assimilated nicotinamide and pantothenic acid accumulated in cofactor forms. Despite the obvious uptake of pyridoxine, accumulation of B₆ vitamers was not observed. In contrast with the other vitamins studied, riboflavin began accumulating in the culture medium immediately after vitamin addition was initiated. By the end of the experiment, the apparent uptake of all vitamins exceeded their accumulation in the cells. Variations in the growth rate of yeast at different vitamin dosages demonstrate the importance of balancing the vitamins in the media during cultivation.     

A model for beta-galactosidase production with a recombinant yeast Saccharomyces cerevisiae in fed-batch culture.

An unstructured model is developed to describe the growth and product formation behavior of a recombinant yeast Saccharomyces cerevisiae using beta-galactosidase as a model protein. The model shows good agreement with the experimental data over a range of conditions. It also accurately predicts the effect of growth rate on yield coefficient and gene expression. The simplicity and accuracy of the model make it suitable for designing and implementing control and optimization strategies for the production of recombinant proteins at large scale.     

Stochastic models to study the impact of mixing on a fed-batch culture of Saccharomyces cerevisiae

The mechanisms of interaction between microorganisms and their environment in a stirred bioreactor can be modeled by a stochastic approach. The procedure comprises two submodels: a classical stochastic model for the microbial cell circulation and a Markov chain model for the concentration gradient calculus. The advantage lies in the fact that the core of each submodel, i.e., the transition matrix (which contains the probabilities to shift from a perfectly mixed compartment to another in the bioreactor representation), is identical for the two cases. That means that both the particle circulation and fluid mixing process can be analyzed by use of the same modeling basis. This assumption has been validated by performing inert tracer (NaCl) and stained yeast cells dispersion experiments that have shown good agreement with simulation results. The stochastic model has been used to define a characteristic concentration profile experienced by the microorganisms during a fermentation test performed in a scale-down reactor. The concentration profiles obtained in this way can explain the scale-down effect in the case of a Saccharomyces cerevisiae fed-batch process. The simulation results are analyzed in order to give some explanations about the effect of the substrate fluctuation dynamics on S. cerevisiae.     

13C-Labeled metabolic flux analysis of a fed-batch culture of elutriated Saccharomyces cerevisiae

This study addresses the question of whether observable changes in fluxes in the primary carbon metabolism of Saccharomyces cerevisiae occur between the different phases of the cell division cycle. To detect such changes by metabolic flux analysis, a 13C-labeling experiment was performed with a fed-batch culture inoculated with a partially synchronized cell population obtained through centrifugal elutriation. Such a culture exhibits dynamic changes in the fractions of cells in different cell cycle phases over time. The mass isotopomer distributions of free intracellular metabolites in central carbon metabolism were measured by liquid chromatography-mass spectrometry. For four time points during the culture, these distributions were used to obtain the best estimates for the metabolic fluxes. The obtained flux fits suggested that the optimally fitted split ratio for the pentose phosphate pathway changed by almost a factor of 2 up and down around a value of 0.27 during the experiment. Statistical analysis revealed that some of the fitted flux distributions for different time points were significantly different from each other, indicating that cell cycle-dependent variations in cytosolic metabolic fluxes indeed occurred.     

Genome-scale analysis of Saccharomyces cerevisiae metabolism and ethanol production in fed-batch culture

A dynamic flux balance model based on a genome-scale metabolic network reconstruction is developed for in silico analysis of Saccharomyces cerevisiae metabolism and ethanol production in fed-batch culture. Metabolic engineering strategies previously identified for their enhanced steady-state biomass and/or ethanol yields are evaluated for fed-batch performance in glucose and glucose/xylose media. Dynamic analysis is shown to provide a single quantitative measure of fed-batch ethanol productivity that explicitly handles the possible tradeoff between the biomass and ethanol yields. Productivity optimization conducted to rank achievable fed-batch performance demonstrates that the genetic manipulation strategy and the fed-batch operating policy should be considered simultaneously. A library of candidate gene insertions is assembled and directly screened for their achievable ethanol productivity in fed-batch culture. A number of novel gene insertions with ethanol productivities identical to the best metabolic engineering strategies reported in previous studies are identified, thereby providing additional targets for experimental evaluation. The top performing gene insertions were substrate dependent, with the highest ranked insertions for glucose media yielding suboptimal performance in glucose/xylose media. The analysis results suggest that enhancements in biomass yield are most beneficial for the enhancement of fed-batch ethanol productivity by recombinant xylose utilizing yeast strains. We conclude that steady-state flux balance analysis is not sufficient to predict fed-batch performance and that the media, genetic manipulations, and fed-batch operating policy should be considered simultaneously to achieve optimal metabolite productivity.     

Improvement of extracellular recombinant glucose oxidase production in fed-batch culture of Saccharomyces cerevisiae: Effect of different feeding strategies

Out of four different feeding strategies tested for the production of extracellular recombinant glucose oxidase from Saccharomyces cerevisiae, constant feeding of galactose on the exhaustion of initial glucose, gave the highest yield-154 U/ml which was 62% above the yield achieved in batch operation (95 U/ml). © Rapid Science Ltd. 1998     

Cysteine addition strategy for maximum glutathione production in fed-batch culture of Saccharomyces cerevisiae

An efficient method of growing the protozoon Tetrahymena to high cell densities in a 2-1 bioreactor is described. The first phase of the fermentation is a batch phase with minimum generation times (1.4 h). During the next phase medium is exchanged continuously using a perfusion module based on microporous hollow fibres while cell are retained. Compared to standard batch fermentations of this organism 30- to 40-fold higher cell concentrations and dry weights were achieved routinely. A maximum cell concentration of 2.2 × 10⁷ cells/ml and a dry weight of 54 g/l have been obtained. As estimated from isocitrate dehydrogenase activity in the culture medium, no cell damage occurred even at high agitation rates. In addition, the cells remained viable for several weeks. Temporal limitation of the process was due to a decrease in the perfusion rate caused by blocking of the membranes. By X-ray microprobe analysis calcium phosphate depositions were detected in the pores of the clogged hollow-fibre membranes. However, even a T. pyriformis strain possessing mucocysts, dense core secretory organelles that may lead to early membrane clogging, was cultivated successfully for 3 weeks. Additionally, the consumption of nutrient protein and carbohydrates during fermentation was investigated and the effect of different perfusion rates and of glucose was studied in order to increase the efficieny of the system.Correspondence to: A. Tiedtke     

Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process

Several bottlenecks in the alcoholic fermentation process must be overcome to reach a very high and competitive performance of bioethanol production by the yeast Saccharomyces cerevisiae. In this paper, a nutritional strategy is described that allowed S. cerevisiae to produce a final ethanol titre of 19% (v/v) ethanol in 45 h in a fed-batch culture at 30 degrees C. This performance was achieved by implementing exponential feeding of vitamins throughout the fermentation process. In comparison to an initial addition of a vitamin cocktail, an increase in the amount of vitamins and an exponential vitamin feeding strategy improved the final ethanol titre from 126 g l(-1) to 135 g l(-1) and 147 g l(-1), respectively. A maximum instantaneous productivity of 9.5 g l(-1) h(-1) was reached in the best fermentation. These performances resulted from improvements in growth, the specific ethanol production rate, and the concentration of viable cells in response to the nutritional strategy.     

On-line adaptive control of a fed-batch fermentation of Saccharomyces cerevisiae

The feasibility of applying an adaptive control technique to a fermentation process is investigated. The nonlinear, time-variant parameters of a fermentation process were estimated on-line as a series of linearized describing matrices. The matrices were used to update a suboptimal feedback law which controlled the process in real time over the linear region. Experiments were performed on a small-scale fully instrumented fermenter with the online, real-time adaptive control package. Results are presented for both single- and multivariable control, and indicate successful control of yeast cell growth.     

Effect of nitrogen limitation on the ergosterol production by fed-batch culture of Saccharomyces cerevisiae

The diversity and content of available nitrogen sources in the growth medium both are very important in the accumulation of ergosterol in the yeast cell membrane. Growth on the good nitrogen sources such as ammonia can harvest more yeast cells than on poor ones, but ergosterol content in those yeast cells is relatively lower. Ergosterol content, one of the most variable parameters in ergosterol production by yeast cultivation, is greatly influenced by nitrogen limitation. The aim of our work was to study how the nitrogen sources affected the membrane ergosterol content and increase the total ergosterol yield. On the premise of keeping high ergosterol content in yeast cell, the ergosterol yield was enhanced by increasing the yeast biomass. Direct feed back control of glucose using an on-line ethanol concentration monitor was introduced to achieve high cell density. Ammonia, which acted as nitrogen source, was added to adjust pH during fermentation process, but its addition needed careful control. Cultivation in 5 L bioreactor was carried out under following conditions: culture temperature 30+/-1 degrees C, pH 5.5+/-0.1, agitation speed 600 rpm, controlling ethanol concentration below 1% and controlling ammonium ion concentration below 0.1 mol/L. Under these conditions the yeast dry weight reached 95.0+/-2.6 g/L and the ergosterol yield reached 1981+/-34 mg/L.     

Calorimetric control of fed-batch cultures of Saccharomyces cerevisiae

Calorimetry has been used to control the glucose feeding in fed-batch cultures of S. cerevisiae in order to avoid ethanol formation and maintain a fully respiratory metabolism. Comparisons between batch and fed-batch cultivations showed that the former had a much lower growth yield. The growth yields for fed-batch cultivations were more than 30% higher than for batch cultures. However, energy balance calculations showed that a large part of the increase could be explained by the evaporation of ethanol during batch cultivations. When the growth yields obtained from the batch cultures were corrected for the evaporation of ethanol, the increase in growth yield for fed-batch cultures was about 10%.     

Influence of bioreactor hydraulic characteristics on a Saccharomyces cerevisiae fed-batch culture: hydrodynamic modelling and scale-down investigations

Yeast is a widely used microorganism at the industrial level because of its biomass and metabolite production capabilities. However, due to its sensitivity to the glucose effect, problems occur during scale-up to the industrial scale. Hydrodynamic conditions are not ideal in large-scale bioreactors, and glucose concentration gradients can arise when these bioreactors are operating in fed-batch mode. We have studied the effects of such gradients in a scale-down reactor, which consists of a mixed part linked to a non-mixed part by a recirculation pump, in order to mimic the hydrodynamic conditions encountered at the large scale. During the fermentation tests in the scale-down reactor, there was a drop in both biomass yield (ratio between the biomass produced and the glucose added) and trehalose production and an increase in both fermentation time (time between inoculation and beginning of stationary phase) and ethanol production. We have developed a stochastic model which explains these effects as the result of an induction process determined mainly by the hydrodynamic conditions. The concentration profiles experienced by the microorganisms during the scale-down tests were expressed and linked to the biomass yields of the scale-down tests.     

Effect of galactose feeding on the improved production of hirudin in fed-batch cultures of recombinant Saccharomyces cerevisiae

Different feeding strategies of galactose were employed to improve the production of anticoagulant, hirudin, by fed-batch mode of cultivation from recombinant Saccharomyces cerevisiae. The structural gene coding for hirudin was harboured with GAL10 promoter for controlled expression of hirudin and the MFα 1 signal sequence for secretion into the growth medium. A step-wise feeding of galactose was found as more suitable feeding strategy of galactose which resulted in the final hirudin volumetric productivity of 6,840?μg/l?·?h, than intermittent, continuous and ethanol controlled feeding of galactose. The final volumetric productivity of hirudin obtained by step-wise feeding of galactose was 3.88 fold higher compared with simple batch fermentation.     

Enhancement of extracellular glucose oxidase production in pH-stat feed-back controlled fed-batch culture of recombinant Saccharomyces cerevisiae

Glucose oxidase (GOD) from Aspergillus niger was expressed in Saccharomyces cerevisiae under the regime of GAL-10 promoter and GAL-7 terminator of S. cerevisiae and -amylase signal sequence of Aspergillus oryzae. The enhancement of the expression level was achieved in pH-stat feed-back controlled fed-batch culture. The highest titre of extracellular GOD was 199 U/ml which marked two fold improvement over the batch (95 U/ml) and 28% above that of non-feed back controlled fed-batch (154 U/ml) operation. © Rapid Science Ltd. 1998     

High-level production of ethanol during fed-batch ethanol fermentation with a controlled aeration rate and non-sterile glucose powder feeding of Saccharomyces cerevisiae

In this study, we utilized a unique strategy for fed-batch fermentation using ethanol-tolerant Saccharomyces cerevisiae to achieve a high-level of ethanol production that could be practically applied on an industrial scale. During this study, the aeration rate was controlled at 0.0, 0.13, 0.33, and 0.8 vvm to determine the optimal aeration conditions for the production of ethanol. Additionally, non-sterile glucose powder was fed during fed-batch ethanol fermentation and corn-steep liquor (CSL) in the medium was used as an organic N-source. When aeration was conducted, the ethanol production and productivity were superior to that when aeration was not conducted. Specifically, the maximum ethanol production reached approximately 160 g/L, when the fermentor was aerated at 0.13 vvm. These findings indicate that the use of a much less expensive C-source may enable the fermentation process to be directed towards the improvement of overall ethanol production and productivity in fermentors that are aerated at 0.13 vvm. Furthermore, if a repeated fed-batch process in which the withdrawal and fill is conducted prior to 36 h can be employed, aeration at a rate of 0.33 and/or 0.8 vvm may improve the overall ethanol productivity     

Glutathione excretion in response to heterologous protein secretion in Saccharomyces cerevisiae

Glutathione is excreted in a dose-dependent, non-stoichiometric fashion from Saccharomyces cerevisiae cells expressing and secreting Bovine Pancreatic Trypsin Inhibitor (BPTI), a small, disulfide-bonded protein. Glutathione excretion commences 40 hours following induction of BPTI synthesis. Expression of several secretory proteins with varying disulfide and cysteine contents results in glutathione excretion with no apparent requirement for protein disulfide content. Glutathione excretion is also triggered by overexpression of Kar2p/BiP, a native ER-resident protein-folding chaperone, indicating that the response is a general one not restricted to overexpression of thiol-containing heterologous proteins. Functional vesicular transport is not required at the time of glutathione excretion, and glutathione excretion requires the presence of molecular oxygen. These data are consistent with a delayed oxidative stress response potentiated by earlier heterologous secretion, but are inconsistent with secretory transport of glutathione spent as oxidizing equivalents for disulfide-bond formation in the endoplasmic reticulum.     

Effect of feed zone in fed-batch fermentations of Saccharomyces cerevisiae

In production-scale, fed-batch fermentations, feed is often added to a single point at the top of the fermentor, which, combined with poor mixing, results in formation of a "feed zone" rich in nutrients. Frequent exposure of the culture to high concentrations of nutrients in the feed zone for sufficient duration can produce unexpected effects on its performance. The effect of the feed zone was evaluated by conducting aerobic fed-batch fermentations of Saccharomyces cerevisiae with both complex and defined media. The broth was recirculated between a recycle loop and a bench-scale fermentor, and feed was intermittently added into the recycle loop to simulate the circulation of cells through the feed zone. Experiments were carried out for a range of residence times in the recycle loop from 0.5 to 12 min. Biomass yields from the complex-media fermentations were not affected by exposure to high nutrient levels in the recycle loop for residence times up to 12 min. Ethanol consumption was reduced by as much as 50% for residence time in the loop up to 3 min. Very long exposure of yeast cells to excess nutrient levels (12 min) gave acetic acid formation. In a defined medium, the simulated feed zone effect increased biomass yield by up to 10%, but had no effect on ethanol levels. This study indicates that the feed zone effect on biomass yield in yeast fermentation, using complex substrates, will be negligible under fully aerobic conditions.     

Cystathionine accumulation in Saccharomyces cerevisiae.

A cysteine-dependent strain of Saccharomyces cerevisiae and its prototrophic revertants accumulated cystathionine in cells. The cystathionine accumulation was caused by a single mutation having a high incidence of gene conversion. The mutation was designated cys3 and was shown to cause loss of gamma-cystathionase activity. Cysteine dependence of the initial strain was determined by two linked and interacting mutations, cys3 and cys1 . Since cys1 mutations cause a loss of serine acetyltransferase activity, our observation led to the conclusion that S. cerevisiae synthesizes cysteine by sulfhydrylation of serine with hydrogen sulfide and by cleavage of cystathionine which is synthesized from serine and homocysteine.     

Enhancement of plasmid stability and protein productivity using multi-pulse, fed-batch culture of recombinant Saccharomyces cerevisiae

The plasmid stability of a recombinant Saccharomyces cerevisiae strain, which expresses cloned -amylase, was increased when glucose or yeast extract was fed with multi-pulse mode in fed-batch culture. Using a novel strategy combining constant rate fed-batch culture and multi-pulse feeding of yeast extract, the plasmid stability was maintained over 90%, meanwhile, 36 g cells l^–1 and 208 units of cloned -amylase activity ml^–1 were obtained.     

Glycerol production by semicontinuous fed-batch fermentation with immobilized cells of Saccharomyces cerevisiae

Summary Semicontinuous fed-batch glycerol fermentations with Saccharomyces cerevisiae cells immobilized in sintered glass Raschig rings were carried out in fixed-bed loop reactors with a working volume of either 0.8 l or 8 l. The influence of biomass, temperature and CO₂ gassing on the glycerol yield was examined. The highest glycerol yield of 85 g l^–1 was achieved at 30° C and average CO₂ gassing rate of 0.4 v/v m with a theoretical glycerol yield of 67%. Fed-batch fermentations with free cells indicated an inhibition mechanism of the glycerol produced, affecting the fermentation capacity of the yeast strain used.Offprint requests to: H.-J. Rehm