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

A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-associated 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 0.12-0.18 h-1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fed-batch 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 formation 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 1-1 culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth.     

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.     

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     

Regulation of proteinase synthesis in Lactococcus lactis

The synthesis of extracellular serine proteinase of Lactococcus lactis was studied during the growth in a batch and a continuous culture on chemically defined media. In a batch culture the proteinase synthesis started during the exponential phase of growth and the highest proteinase concentrations were found at the end of the exponential and beginning of the stationary phase of growth. During the growth in a lactose-limited chemostat with amino acids as the sole source of nitrogen, the specific rate of proteinase synthesis was maximal at a μof 0.23 h^?1. At higher growth rates the proteinase productin declined. The proteinase synthesis was dependent on the amino acid sources in the medium. In batch cultures of L. lactis grown on a chemically defined medium with amino acids, the proteinase production was increased four-fold compared to media containing casein or a tryptic digest of casein as the sole source of nitrogen. The inhibition of the rate of proteinase synthesis by casein and peptides was also observed during the growth in a chemostat. The addition of the dipeptide leucylproline (final concentration of 100 μM) to a lactose-limited continuous culture during the steady state (D = 0.23 h^?1) resulted in a transient inhibition of the rate of proteinase synthesis. This suggested that exogenously supplied peptides control the regulation of proteinase synthesis of L. lactis.     

Influence of culture modes on the microbial production of hyaluronic acid by <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

The principal objective of this study was to assess the effects of culture modes including batch culture, pulse fed-batch culture, constant feeding rate fed-batch culture, and exponential fed-batch culture on the production of hyaluronic acid (HA) by Streptococcus zooepidemicus. Batch cultures had the highest levels of HA productivity, whereas fed-batch cultures were more favorable with regard to cell growth, and exponential fed-batch cultures evidenced the highest cell concentrations. A two-step culture model was proposed to enhance HA production: an exponential fed-batch culture was conducted prior to 8 h and then sucrose supplementation was applied for 8 h to start the batch fermentation of S. zooepidemicus. HA production and productivity were increased by 36 and 37% in the proposed two-step culture process as compared with that observed in the batch culture, respectively. The proposed two-step culture model can be applied in the production of secondary metabolites, and particularly of the exopolysaccharides.     

Effect of an exponential feeding regime on the production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica

Aims: To evaluate the effect of and exponential feeding regime on the production of epoxide hydrolase (EH) enzyme in recombinant Yarrowia lipolytica in comparison to a constant feed strategy. Methods and Results: An exponential feed model was developed and fermentations were fed at six different exponential rates. A twofold increase in EH productivity and a 15% increase in volumetric EH activity was obtained by applying exponential glucose feed rates in fed‐batch cultivation. These responses were modelled to obtain a theoretical optimum feed rate that was validated in duplicate fermentations. The model optimum of 0·06 h^?1 resulted in a volumetric EH activity of c. 5500 U l^?1 h^?1 and a maximum activity of 206 000 U l^?1. This correlated well with model predictions, with a variance of <10%. Conclusions: The use of an exponential feed strategy at a rate of 0·06 h ^{? 1} yielded best results for all key responses which show a clear improvement over a constant feed strategy. Significance and Impact of the Study: The study was the first evaluation of an exponential feed strategy on recombinant Y. lipolytica for the production of EH enzyme. The results suggest a strategy for the commercial production of a valuable pharmaceutical enzyme.     

Open-loop control of specific growth rate in fed-batch cultures of recombinant E.coli

Summary A computer-based algorithm was used for the open-loop control of specific growth rate in fed-batch cultures of recombinant E.coli.. The control of nutrient feed rate to an exponential trajectory resulted in growth of the culture at a constant specific growth rate. Stable specific growth rates between 0.08 and 0.4 h^–1 were achieved.     

Redirection of metabolism during nutrient feeding in fed-batch cultures of Bacillus thuringiensis

During sporulation, Bacillus thuringiensis produces insecticidal crystal inclusions (Cry proteins) encoded by cry genes. In fed-batch cultures (FBCs), spores and Cry protein yields are usually low, so we therefore studied the pattern of metabolic changes occurring in batch cultures and FBCs of a B. thuringiensis strain having a cry1Aa promoter-lacZ fusion, and their effect on sporulation and cry1A gene expression. In FBCs, there was a redirection of bacterial metabolism and a reduction in the specific growth rate during feeding, even when the nutrient concentration was higher than at the beginning of batch culture. These physiological changes suggest that the transition state is set up during feeding and this set-up seems to have a negative effect on both sporulation and cry1Aa expression. When the filtrate of a culture in the transition state was added to a batch culture early in the first exponential growth phase, it delayed sporulation and cry1Aa expression, thus suggesting that a soluble cellular factor that blocked sporulation might be excreted during the transition state. Citrate production usually started during the transition state but, when a medium rich in free amino acids was fed, citrate was produced from the first growth phase and sporulation was nearly blocked.     

Cell-mass maximization in fed-batch cultures

Complete solutions are provided for cell-mass maximization for free and fixed final times and constant and variable yields. The optimal feed rate profile is a concatenation of maximum, minimum and singular feed rates. The exact sequence and duration of each feed rate depends primarily on the initial substrate concentration, and degenerate cases arise due to the magnitude constraint on the feed rate and the length of final time t _f. When the final time is free and not in the performance index, it is infinite for constant yield so that any form of feed rate leads to the same amount of cells, while for variable yield the singular feed rate is exponential and maximizes the yield. For fixed final time the singular feed rate for constant yield is exponential and maximizes the specific growth rate by maintaining the substrate concentration constant, while for variable yield, it is semi-exponential and the substrate concentration starts near the maximum specific growth rate and moves toward the maximum yield. A simple sufficient condition for existence of singular feed rate requires an existence of a region bounded by the maxima of specific growth and cellular yield. Otherwise, the optimal feed rate profile is a bang-bang type and the bioreactor operates in batch mode.     

Increased Hepatitis B surface antigen production by recombinant <Emphasis Type="BoldItalic">Aspergillus niger</Emphasis> through the optimization of agitation and dissolved oxygen concentration

The capacity of the filamentous fungi Aspergillus niger to produce and assemble complex immunogenic viral proteins into virus-like particles (VLPs) in batch culture was enhanced by optimizing the bioprocessing parameters, agitation intensity and dissolved oxygen (dO₂) concentration. Response surface methodology (RSM) and a two-factor-two-level central composite rotatable design (CCRD) were employed to evaluate the interactive response pattern between parameters and their optimum combination. The recombinant hepatitis B surface antigen (HBsAg) was used as a model VLP system to determine the effect of these parameters on biomass yield, fungal morphology, HBsAg production and bioreactor kinetics. The response surface model predicted optimum cultivation conditions at an agitation of rate of 100 rpm and a dO₂ concentration of 25%, obtaining highest intracellular membrane-associated HBsAg levels of . HBsAg production levels were increased tenfold compared to yields obtained in shake flask cultivation. Although hepatitis B VLPs mostly accumulated intracellularly, optimal bioreactor conditions resulted in significant HBsAg release in culture supernatant. These results compare favourably with other recombinant VLP systems in batch culture, and therefore, indicate a substantial potential for further engineering of the A. niger production system for the high level of intracellular and extracellular VLP production.     

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.     

Effect of urea on the production of 2,3-butanediol by K. oxytoca

This paper deals with the production of 2,3- butanediol by K. oxytoca in batch cultures. The effect of urea on various kinetic parameters was studied by replacing the ammonium salts in the medium with the corresponding nitrogen equivalent in the form of urea. The specific growth rate and the product yield in an unacclimatised batch culture were found to be 0.29 h^?1 and 0.26 g·g^?1 respectively. The acclimatised batch cultures on the other hand behaved similar to that grown using the original medium with a specific growth rate of 0.66 h^?1 and the product yield of 0.345 g·g^?1. However the cultures were unable to grow when urea was used both as the carbon and nitrogen source.     

The activity of beta-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate

Aims: Kluyveromyces lactis was cultured in cheese whey permeate on both batch and continuous mode to investigate the effect of time course and growth rate on β‐galactosidase activity, lactose consumption, ethanol production and protein profiles of the cells. Methods and Results: Cheese whey was the substrate to grow K. lactis as a batch or continuous culture. In order to precise the specific growth rate for maximum β‐galactosidase activity a continuous culture was performed at five dilution (growth) rates ranging from 0·06, 0·09, 0·12, 0·18 to 0·24 h^?1. The kinetics of lactose consumption and ethanol production were also evaluated. On both batch and continuous culture a respirofermentative metabolism was detected. The growth stage for maximum β‐gal activity was found to be at the transition between late exponential and entrance of stationary growth phase of batch cultures. Fractionating that transition stage in several growth rates at continuous culture a maximum β‐galactosidase activity at 0·24 h^?1 was observed. Following that stage β‐gal activity undergoes a decline which does not correlate to the density of its corresponding protein band on the gel prepared from the same samples. Conclusion: The maximum β‐galactosidase activity per unit of cell mass was found to be 341·18 mmol ONP min^?1 g^?1 at a dilution rate of 0·24 h^?1. Significance and Impact of the Study: The physiology of K. lactis growing in cheese whey permeate can proven useful to optimize the conversion of that substrate in biomass rich in β‐gal or in ethanol fuel. In addition to increasing the native enzyme the conditions established here can be set to increase yields of recombinant protein production based on the LAC4 promoter in K. lactis host.     

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.     

Kinetics of α-amylase production in a batch and a fed-batch culture ofBacillus subtilis

Analysis of the kinetics of α-amylase production in a batch and a fed-batch culture ofBacillus subtilis made it possible to derive a konetic model of the process describing mutual interactions between growth and production. The specific growth rate is limited by the concentration of both corn-steep liquor and starch. Higher concentrations of reducing sugars in the medium also inhibit growth. The overall production of α-amylase is a result of an equilibrium between the rate of enzyme production and its degradation due to the effect of environment. The actual specific production rate is directly proportional to the specific production rate is directly proportional to the specific growth rate (characterizing the physiological state of the culture) and is inhibited by higher concentrations of corn-steep liquor in the medium.     

Elicitation with methyl-jasmonate stimulates peruvoside production in cell suspension cultures of <Emphasis Type="Italic">Thevetia peruviana</Emphasis>

Thevetia peruviana is a small tree that produces several compounds with pharmaceutical application, among which peruvoside could be highlighted. However, these compounds are produced in low concentration in the plant, making it important to develop strategies such as plant cell culture and elicitation to obtain higher quantities of the desired product. In this work, cell suspension cultures of T. peruviana were established in four different culture media: Murashige–Skoog (MS), half Murashige–Skoog (half MS), Schenk–Hildebrandt (SH), and Gamborg (B5) to study their effect on cell growth. Cell growth kinetics were studied in SH medium, and the extracellular peruvoside production during the culture time was determined. The best culture medium for the establishment of cell suspension cultures was MS with a growth index of 3.17 ± 0.2 g g⁻¹ inoculum. The cell growth kinetics showed the four characteristic growth phases of a cell culture (lag, exponential, stationary, and death), and during none of these phases was it possible to observe peruvoside production. The elicitor effect of methyl-jasmonate (MeJ) was studied in cell suspension cultures established in SH medium. The effect of MeJ concentration and the time in which it should be applied were determined. The best results were obtained at a concentration of 100 mg l⁻¹ of MeJ applied at the beginning of the culture, which induced a peruvoside production of 8.93 mg l⁻¹ medium. The current results are the first report of an in vitro peruvoside production system.     

Influence of Environmental Factors and Medium Composition on Vibrio gazogenes Growth and Prodigiosin Production

Vibrio gazogenes ATCC 29988 growth and prodigiosin synthesis were studied in batch culture on complex and defined media and in chemostat cultures on defined medium. In batch culture on complex medium, a maximum growth rate of 0.75 h⁻¹ and a maximum prodigiosin concentration of 80 ng of prodigiosin · mg of cell protein⁻¹ were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h⁻¹), and maximum prodigiosin concentrations were higher (1,500 ng · mg of protein⁻¹). In batch culture on either complex or defined medium, growth was characterized by a period of logarithmic growth followed by a period of linear growth; on either medium, prodigiosin biosynthesis was maximum during linear growth. In batch culture on defined medium, the initial concentration of glucose optimal for growth and pigment production was 3.0%; higher levels of glucose suppressed synthesis of the pigment. V. gazogenes had an absolute requirement for Na⁺; optimal growth occurred in the presence of 100 mM NaCl. Increases in the concentration of Na⁺ up to 600 mM resulted in further increases in the concentration of pigment in the broth. Prodigiosin was synthesized at a maximum level in the presence of inorganic phosphate concentrations suboptimal for growth. Concentrations of KH₂PO₄ above 0.4 mM caused decreased pigment synthesis, whereas maximum cell growth occurred at 1.0 mM. Optimal growth and pigment production occurred in the presence of 8 to 16 mg of ferric ion · liter⁻¹, with higher concentrations proving inhibitory to both growth and pigment production. Both growth and pigment production were found to decrease with increased concentrations of p-aminobenzoic acid. The highest specific concentration of prodigiosin (3,480 ng · mg protein⁻¹) was observed in chemostat cultures at a dilution rate of 0.057 h⁻¹. The specific rate of prodigiosin production at this dilution rate was approximately 80% greater than that observed in batch culture on defined medium. At dilution rates greater than 0.057 h⁻¹, the concentration of cells decreased with increasing dilution rate, resulting in a profile comparable to that expected for linear growth kinetics. No explanation could be found for the linear growth profiles obtained for both batch and chemostat cultures.     

Growth and development kinetics of Bacillus thuringiensis in batch culture

The kinetics of Bacillus thuringiensis growth and its assimilation of nutrient substances were studied under the conditions of batch cultivation in a complex medium containing yeast extract and in a chemically defined medium with amino acids. The growth of B. thuringiensis can be divided into five phases: exponential growth; decelerated growth; stationary phase when protein crystals are formed; stationary phase when spores are formed; lysis of sporangia releasing spores. The first phase may in turn be subdivided into three stages according to changes in the specific growth rate and substrate assimilation: a high specific growth rate and no glucose assimilation; an abrupt drop in mu and the beginning of intensive glucose assimilation from the medium; a new rise in the specific growth rate. As follows from the results of studying the kinetics of B. thuringiensis growth in a chemically defined medium, the above changes in the exponential growth phase are due to the fact that the culture assimilates yeast extract components in the complex medium or amino acids in the chemically defined medium during this phase, and then starts to assimilate glucose and ammonium in the following phases of growth.     

Optimization of glucose feeding approaches for enhanced glucosamine and N-acetylglucosamine production by an engineered Escherichia coli

In this work, a recombinant Escherichia coli was constructed by overexpressing glucosamine (GlcN) synthase and GlcN-6-P N-acetyltransferase for highly efficient production of GlcN and N-acetylglucosamine (GlcNAc). For further enhancement of GlcN and GlcNAc production, the effects of different glucose feeding strategies including constant-rate feeding, interval feeding, and exponential feeding on GlcN and GlcNAc production were investigated. The results indicated that exponential feeding resulted in relatively high cell growth rate and low acetate formation rate, while constant feeding contributed to the highest specific GlcN and GlcNAc production rate. Based on this, a multistage glucose supply approach was proposed to enhance GlcN and GlcNAc production. In the first stage (0–2 h), batch culture with initial glucose concentration of 27 g/l was conducted, whereas the second culture stage (2–10 h) was performed with exponential feeding at μ _set = 0.20 h⁻¹, followed by feeding concentrated glucose (300 g/l) at constant rate of 32 ml/h in the third stage (10–16 h). With this time-variant glucose feeding strategy, the total GlcN and GlcNAc yield reached 69.66 g/l, which was enhanced by 1.59-fold in comparison with that of batch culture with the same total glucose concentration. The time-dependent glucose feeding approach developed here may be useful for production of other fine chemicals by recombinant E. coli.     

Protease and lipase production by a strain ofSerratia marcescens (532 S)

Summary Production of both exolipase and exoprotease activities bySerratia marcescens 532 S isolated from an aerobic fixed-biomass reactor were strongly influenced by nutritional factors which acted as inducers or repressors. In batch culture, protease and lipase activities were produced after the exponential phase. NH₄Cl, amino acids and simple carbon sources caused repression of protease activity. At a concentration of 1.5 g L^–1, the individual addition of maltose, mannitol, acetate, fructose or glucose, repressed exoprotease production, with the greatest effect by glucose. An inverse relationship existed between exoprotease synthesis and increasing glucose concentrations. Lipids activated lipase production, the most significant increase occurred when Tween 80 was added in the medium. Thus, glucidolytic, proteolytic and lipolytic activities could be efficiently expressed in batch cultures only successively.At low dilution rate of chemostat cultures with a constant glucose input concentration of 2 g L^–1, glucidolytic, proteolytic and lipolytic activities were produced, but did not have the same regulation: atD values <0.08 h^–1, the level of protease activity dropped while that of lipase showed a corresponding increase. Above these values, increasingD led to a decrease of the two hydrolase activities, at the level of the specific activities as well as in the specific rate of biosynthesis of each enzyme. Similar results were obtained in chemostat culture with a constant specific growth rate of 0.04 h^–1 with increasing glucose input concentrations, i.e. protease and lipase activities decreased when the specific glucose uptake rates were enhanced.