Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg)

Summary A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-assciated product formation. By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 1.12-0.18 h^–1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fedbatch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture. The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product fromation kinetics. In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-l culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth. Offprint requests to: M. B. Gu     

Production of HBsAg by growth rate control with recombinantSaccharomyces cerevisiae in fed-batch

Summary To maintain a constant specific growth rate for a recombinantS.cerevisiae in fed-batch, the medium feeding rate has been controlled with respect to the hourly calculated glucose uptake rate. The recombinant yeast producing HBsAg showed the exponential production trend in proportion to the exponential cell growth. Total cell yield in fed-batch was about 0.402 g cells/g glucose, and HBsAg was produced about ten times more than in batch. Decrease of growth rate by HBsAg produced was not shown.     

Production of savinase and population viability of Bacillus clausii during high-cell-density fed-batch cultivations

The growth and product formation of a Savinase-producing Bacillus clausii were investigated in high-cell-density fed-batch cultivations with both linear and exponential feed profiles. The highest specific productivity of Savinase was observed shortly after the end of the initial batch phase for all feed profiles applied and, in addition, there was a time-dependent decrease in specific productivity. The specific glucose uptake rate increased with time for constant specific growth rate indicating that the maintenance requirements increased with time, possibly due to a decreasing K(+) concentration. The physiological state of the cells was monitored during the cultivations using a flow cytometry assay based on the permeability of the cell membrane to propidium iodide. In the latter parts of the fed-batch cultures with a linear feed profile, a large portion of the cell population was found to have a permeable membrane, indicating a large percentage of dead cells. By assuming that only cells with a nonpermeable membrane contributed to growth and product formation, the physiological properties of this subpopulation were calculated.     

Effects of high cell density on growth-associated monoclonal antibody production by hybridoma T0405 cells immobilized in macroporous cellulose carriers

Relationship between monoclonal antibody (MAb) productivity and growth rate, and effects of high cell density on MAb production of hybridoma T0405 cells immobilized in macroporous cellulose carriers were investigated in continuous and batch cultures. The results showing, that the specific MAb production rate increased with increasing specific growth rate in both suspended and immobilized continuous cultures indicate a positively growth-associated relationship between MAb productivity and growth rate. Moreover, the specific production rate was higher in the immobilized cell culture than that in suspended one at all dilution rates. In order to clarify these phenomena, MAb mRNA expression and cell cycle distribution were investigated in batch cultures with immobilized cells and suspended cells. RT-PCR was used for observation of MAb mRNA expression and a two-color bromode-oxyuridine (BrdU)/propidium iodide (PI) flow cytometry method for determination of cell cycle distribution. The results revealed that MAb mRNA expression reached the peak during the exponential growth phase, suggest a positively growth-associated MAb production. And the immobilized cells continued the MAb mRNA expression until dead phase, which was longer than that in suspended cells. The cell cycle distribution patterns were observed almost the same for both immobilized and suspended cells. Such results may imply that a high cell density state has positive influence on the mRNA expression and on growth-associated MAb productivity of T0405 cells.     

Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture

Summary Recombinant human interferon- production by Chinese hamster ovary cells was restricted to the growth phase of batch cultures in serum-free medium. The specific interferon production rate was highest during the initial period of exponential growth but declined subsequently in parallel with specific growth rate. This decline in specific growth rate and interferon productivity was associated with a decline in specific metabolic activity as determined by the rate of glucose uptake and the rates of lactate and ammonia production. The ammonia and lactate concentrations that had accumulated by the end of the batch culture were not inhibitory to growth. Glucose was exhausted by the end of the growth phase but increased glucose concentrations did not improve the cell yield or interferon production kinetics. Analysis of amino acid metabolism showed that glutamine and asparagine were exhausted by the end of the growth phase, but supplementation of these amino acids did not improve either cell or product yields. When glutamine was omitted from the growth medium there was no cell proliferation but interferon production occurred, suggesting that recombinant protein production can be uncoupled from cell proliferation. Offprint requests to: P. M. Hayter     

Computer controlled transient state in a fed batch culture

Summary A controlled shift down of the specific growth rate in a fed batch culture is proposed for microbial product formation processes. The growth kinetics depend on three preset parameters: initial biomass concentration, initial specific growth rate, and the transient constant (time derivative of the specific growth rate). An experimental example is given.     

Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin

The study of batch kinetics of Lactococcus lactis cell growth and product formation reveals three distinct metabolic behaviors depending upon the availability of oxygen to the culture and the presence of hemin in the medium. These three cultivation modes, anerobic homolactic fermentation, aerobic heterolactic fermentation, and hemin-stimulated respiration have been studied at pH 6.0 and 30 degrees C with a medium containing a high concentration of glucose (60 g/L). A maximum cell density of 5.78 g/L was obtained in the batch culture under hemin-stimulated respiration conditions, about three times as much as that achieved with anerobic homolactic fermentation (1.87 g/L) and aerobic heterolactic fermentation (1.80 g/L). The maximum specific growth rate was 0.60/h in hemin-stimulated respiration, slightly higher than that achieved in homolactic fermentation (0.56/h) and substantially higher than that in heterolactic fermentation (0.40/h). Alteration of metabolism caused by the supplementation of oxygen and hemin is evidenced by changes in both cell growth kinetics and metabolite formation kinetics, which are characterized by a unique pseudo-diauxic growth of L. lactis. We hypothesise that Lactococcus lactis generates bioenergy (ATP) through simultaneous lactate formation and hemin-stimulated respiration in the primary exponential phase, when glucose is abundant, and utilizes lactate for cell growth and cell maintenance in the stationary phase, after glucose is exhausted. We also examined the applicability of a modified logistic model and the Luedeking-Piret model for cell growth kinetics and metabolite formation kinetics, respectively.     

Kinetics of growth and leukotoxin production by <Emphasis Type="Italic">Mannheimia haemolytica</Emphasis> in continuous culture

The growth and product formation kinetics of the bovine pathogen Mannheimia (Pasteurella) haemolytica strain OVI-1 in continuous culture were investigated. The leukotoxin (LKT) concentration and yield on biomass could substantially be enhanced by supplementation of a carbon-limited medium with an amino acid mixture or a mixture of cysteine and glutamine. Acetic acid was a major product, increasing to 1.66 g l(-1) in carbon-limited chemostat culture at intermediate dilution rates and accounting for more than 80% of the glucose carbon, whereas in amino acid-limited cultures high acetic acid concentrations were produced at low dilution rates, suggesting a carbon-overflow metabolism. The maintenance coefficients of carbon-limited and carbon-sufficient cultures were 0.07 and 0.88 mmol glucose g(-1) h(-1), respectively. LKT production was partially growth-associated and the LKT concentration was maximised to 0.15 g l(-1) and acetic acid production minimised by using a carbon-limited medium and a low dilution rate.     

Defining an optimal carbon source/methionine feed strategy for growth and cephalosporin C formation by Cephalosporium acremonium.

The effect of the method of methionine addition, growth-limiting carbon source (glucose vs sucrose), and culture growth rate on cephalosporin C production was investigated in a Cephalosporium acremonium defined medium fed batch fermentation. Batch addition of methionine, at a concentration of 3 g/L, prior to the start of a fed sucrose fermentation was found to interfere with the ability of the culture to utilize this sugar, thus limiting growth and decreasing cephalosporin C production. Batch methionine addition had no effect on glucose-limited cultures. Concurrent exponential feeding of methionine with sucrose improved both culture growth and productivity. Under the control of identical carbon source limiting feed profiles, sucrose was observed to support greater cephalosporin C production than glucose. Optimal cephalosporin C production in a C. acremonium defined medium fed batch fermentation was obtained through controlling culture growth during the rapid growth phase at a relatively low level with respect to mumax (mu approximately 0.036 h-1) until achieving a desired cell mass with a concurrent sucrose and methionine feed, followed by maintaining relatively vigorous growth (mu approximately 0.01 h-1) with sucrose for the duration of the fermentation.     

Approximation of continuous growth of Cephalotaxus harringtonia plant cell cultures using fed-batch operation

In Cephalotaxus harringtonia plant cell cultures, periods of batch growth that are limited by hexose uptake are too short to make an accurate estimate of the Monod saturation constant. Continuous cultures are infeasible on a laboratory scale, and semicontinuous cultures require too frequent sampling. Fed-batch operation, consisting of intermittent removal from a culture that is fed continuously, was investigated as a possible solution to these problems. For a constant feed rate, computer simulations showed that a steady state can be achieved which is useful for studying growth at different specific growth rates. In terms of the dilution rate it was confirmed that the operation is essentially equivalent to continuous culture when the samples represent a small fraction of the total culture volume. Experiments with glucose or fructose as the carbon source were carried out in shake flasks fed by a multichannel syringe pump. Results indicate that Monod kinetics based on medium glucose levels cannot adequately describe growth under these conditions. Monod's expression for specific growth rate using internal glucose concentration gives an improved correlation.     

Dynamics of l-valine in relation to the production of cyclosporin A by Tolypocladium inflatum

Summary We report the kinetics of endogenous l-valine in the fungus Tolypocladium inflatum, in an effort to understand the enhancing effect of externally supplemented l-valine on the production of the immunosuppressant cyclosporin A (CyA) in chemically defined medium. In a batch laboratory stirred reactor cultivation, the concentration of intracellular l-valine increased by up to four times between the end of the exponential phase and the beginning of the stationary phase when the medium was supplemented externally with 4 g/1 l-valine. The final CyA titre under these conditions was 710 mg/1 compared to only 130 mg/1 attained without l-valine supplementation. In contrast to substantial growth-associated production of CyA in unsupplemented culture, the formation of the immunosuppressant was prolonged during the stationary phase in l-valine-supplemented medium. As a result, the conversion yield of CyA on l-valine remained constant during the stationary phase at 0.27 g CyA/g l-valine.     

Modelling of agrobacteran production in a batch culture ofAgrobacterium radiobacter

A deterministic model of production of the exopolysaccharide agrobacteran, including the effect of the product on oxygen transfer into the medium, was used for evaluation of a batch cultivation ofAgrobacterium radiobacter. Application of mass-energy balance made it possible to reduce the number of identified parameters. The resulting yield coefficients and rate constants were largely independent of the method of aeration of the culture, with the exception of the maximum specific rate of agrobacteran production which was lower if the culture in an exponential growth phase was subjected to oxygen limitation.     

Production of cell mass and pertussis toxin by Bordetella pertussis.

The cultivation of Bordetella pertussis affects production of pertussis toxin and biomass. Comparison of batch mode, chemostat operation and pHstat-turbidostatic control showed that productivities for the continuous process were greater than that for the batch operation. Continuous operation in balanced growth at the maximum specific growth rate, provided by the pHstat, resulted in the maximum specific production rate. Because of the strong association of pertussis toxin synthesis and cell growth, the concentration of toxin in the effluent of the continuous processes was greater than the maximum obtained in the batch bioprocess. An expanded Luedeking-Piret model of product formation kinetics fits the observed chemostat data and demonstrates that the production of pertussis toxin from the culture of B. pertussis is predominantly growth associated.     

Fermentation kinetics of spent sulfite liquor by Saccharomyces cerevisiae

The growth kinetics of the yeast Saccharomyces cerevisiae and the production rate of ethanol have been studied in batch fermentation under anaerobic conditions in a 20-L fermentor. Two substrates were used in fermentation trials: a synthetic mixture of three fermentable sugars, D-glucose, D-mannose, and D-galactose, and a low-yield liquor originating from a bisulfite cooking process. The Monod model adequately described the system in relation to the specific growth rate mu(x) and the specific product formation rate mu(P). Different fermentation parameters (growth rate, substrate utilization, and product formation) were determined for the synthetic mixture and the bisulfite liquor. It was observed that the specific growth rate is much lower in spent sulfite liquor than in a synthetic medium. However, the specific product formation rate remains the same in both media.     

Growth-rate-independent production of recombinant glucoamylase by Fusarium venenatum JeRS 325

Most recombinant proteins generated in filamentous fungi are produced in fed-batch cultures, in which specific growth rate normally decreases progressively with time. Because of this, such cultures are more suited to the production of products that are produced efficiently at low-growth rates (e.g., penicillin) than to products which are produced more efficiently at high-growth rates (e. g., glucoamylase). Fusarium venenatum A3/5 has been transformed (JeRS 325) to produce Aspergillus niger glucoamylase (GAM) under the control of the Fusarium oxysporum trypsin-like protease promoter. No glucoamylase was detected in the culture supernatant during exponential growth of F. venenatum JeRS 325 in batch culture. In glucose-limited chemostat cultures, GAM concentration increased with decrease in dilution rate, but the specific production rate of GAM (g GAM [g biomass](-1) h(-1)) remained approximately constant over the dilution-rate range 0.05 h to 0.19 h(-1), i.e., the recombinant protein was produced in a growth-rate-independent manner. The specific production rate decreased at dilution rates of 0.04 h(-1) and below. Specific production rates of 5.8 mg and 4.0 mg GAM [g biomass](-1) h(-1) were observed in glucose-limited chemostat cultures in the presence and absence of 1 g mycological peptone L(-1). Compared to production in batch culture, and for the same final volume of medium, there was no increase in glucoamylase production when cultures were grown in fed-batch culture. The results suggested that a chemostat operated at a slow dilution rate would be the most productive culture system for enzyme production under this trypsin-like promoter.     

Dialysis fermentation. II. Kinetic analysis of salicylic acid production via cell growth and maintenance

A kinetic analysis was made of the relationship between salicylate production from naphthalene and growth of Pseudomonas fluorescens in semicontinuous dialysis culture. The specific rates both of product formation and growth initially were increased by the diffusional withdrawal of salicylate, but subsequently were reduced to low levels despite continued salicylate removal. Productivity and growth were correlated by the Luedeking-Piret equation in an initial nondialysis period and in the early stages of dialysis fermentation, when specific growth rates exceeded. 005 hr^?1. Below this level of growth at later stages of dialysis fermentation, the specific production rate was correlated only with total cell mass by a proportionality constant of .035 hr^?1, which was attributed to maintenance metabolism. Maintenance accounted for about 84% of the total salicylate produced, while growth-associated metabolism accounted for the remainder.     

Step-up/step-down perfusion approach for increased mAb 520C9 production by a hybridoma cell line

The hybridoma cell line, HB-8696, produces a monoclonal antibody, 520C9 (mouse IgG₁) that recognizes the breast cancer oncoprotein, c-erbB2. The effect of perfusion rate (volume of fresh feed/working volume of reactor/day) on cell growth and mAb production was investigated but perfusion at a constant rate and at an arbitrarily increased rate could not maintain exponential cell growth or a higher specific mAb production rate. An optimum step-up/step-down perfusion strategy is therefore proposed for maintaining a steady state production phase at high cell density for ten days. The optimum step-up perfusion could achieve fast cell growth by avoiding any nutrient limited condition and the following optimum step-down perfusion could potentially maintain high live cell density and reduced product dilution as well. The maximum viable cell achieved under optimum perfusion strategy was 2.3 × 10⁷ cells/ml which was 19-fold higher than in optimum batch culture. The mAb yield and volumetric productivity were significantly improved to 52 and 50 mg/l day compared to 25 and 3.8 mg/l day in optimum batch, respectively, and could be maintained for up to ten days.     

Physiology of the growth and supersynthesis of DNA-polymerase in Escherichia coli during 2-stage continuous cultivation

The physiology of growth under the conditions of batch and continuous cultivation was studied with the recombinant strain of Escherichia coli CM 5199 capable of DNA polymerase I superproduction. The specific growth rate of the strain is 0.8 h-1 under the conditions of continuous cultivation which is almost 2.5 times greater than that in the exponential phase of batch cultivation. When the strain was cultivated at a flow rate above 0.3 h-1, the biomass concentration in the fermenter decreased and the culture was no more limited by the carbon source in the absence of other growth limiting components of the medium. Apparently, the metabolic product ceased to inhibit high growth rates of the culture under the conditions of continuous cultivation. The rate of DNA polymerase synthesis correlated with the specific growth rate and the respiration activity of the culture when the lambda pol A prophage was induced in the cells. The authors discuss the effectiveness of ribosome operation in the cells at a growth rate of 0.05 to 0.3 h-1 and the content of ribosomes at a higher growth rate in relation to DNA polymerase I synthesis.     

Vancomycin production in batch and continuous culture

Production of the glycopeptide antibiotic vancomycin by two Amycolatopsis orientalis strains was examined in batch shake flask culture in a semidefined medium with peptone as the nitrogen source. Different growth and production profiles were observed with the two strains; specific production (Y(p/x)) was threefold higher with strain ATCC 19795 than with strain NCIMB 12945. A defined medium with amino acids as the nitrogen source was developed by use of the Plackett-Burman statistical screening method. This technique identified certain amino acids (glycine, phenylalanine, tyrosine, and arginine) that gave significant increased specific production, whereas phosphate was identified as inhibitory for high specific vancomycin production. Experiments made with the improved medium and strain ATCC 19795 showed that vancomycin production kinetics were either growth dissociated or growth associated, depending on the amino acid concentration. In chemostat culture at a constant dilution rate (0.087 h(-1)), specific vancomycin production rate (q(vancomycin)) decreased linearly as the medium phosphate concentration was increased from 2 to 8 mM. In both phosphate and glucose limited chemostats, q(vancomycin) was a function of specific growth rate; the maximum value was observed at D = 0.087 h(-1) (52% of the maximum specific growth rate). Under phosphate limited growth conditions, q(vancomycin) was threefold higher (0.37 mg/g dry weight/h) than under glucose limitation (0.12 mg/g dry weight/h). (c) 1996 John Wiley & Sons, Inc.