Cybernetic modeling of bacteriol cultures at low growth rates: single-substrate systems

The cybernetic framework developed by Ramkrishna and co-workers has been expanded to include the effects of cellular maintenance energy requirements on biomass levels in slow-growing, carbon-substrate-limited cultures. A simple structured model, based on the existence of distinct key enzymes for growth and maintenance functions, is presented. Comparisons of the model with experimental data for the growth of Klebsiella oxytoca in constant fed-batch culture on glucose, fructose, arabinose, and xylose show good agreement. In addition, perturbed fed-batch culture experiments indicate that slow-growing cultures respond less rapidly to a removal of the growth limitation than do faster-growing ones. The possibility of a growth-rate dependent "critical resource" is discussed.     

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.     

Fermentation kinetics of Zymomonas mobilis near zero growth rate

The fermentation kinetics Zymomonas mobilis were studied near zero growth rate in fed-batch cultures and continuous cultures with complete cell recycle. The results show the ethanol enhances that specific substrate conversion rate under these conditions. The maximum achievable ethanol concentration in continuous cultures with cell recycle (66 g/L) was significantly lower than in fed-batch cultures (100 g/L). The results indicate that growth-rate-independent metabolism is not instantaneous and can lag behind steadily increasing ethanol concentrations in fed-batch fermentations. A model is proposed to account for this slow adaptation.     

Fed-batch culture: Modeling and application in the study of microbiol energetics

Microbial growth in the fed-batch mode is described by a simple unstructured model. The model is found to be in good agreement with agreement with the experimental observation, except under highly transient conditions. Extensive experimental data were collected and the energetics of the bacterium Klebsiella pneumoniae is evaluated. It is shown that the fed-batch culti vation is a powerful experimental tool in the study of microbial kinetics and energetics simultaneously. Methods for determining the maintenance requirements are shown and evaluated. The maintenance coefficients determined from fed-batch data are systematically smaller than those reported for continuous culture systems. Results suggest a decrease in maintenance demands at low specific growth rates.     

Macroscopic modelling of overflow metabolism and model based optimization of hybridoma cell fed-batch cultures

A macroscopic model that takes into account phenomena of overflow metabolism within glycolysis and glutaminolysis is proposed to simulate hybridoma HB-58 cell cultures. The model of central carbon metabolism is reduced to a set of macroscopic reactions. The macroscopic model describes three metabolism states: respiratory metabolism, overflow metabolism and critical metabolism. The model parameters and confidence intervals are obtained via a non linear least squares identification. It is validated with experimental data of fed-batch hybridoma cultures and successfully predicts the dynamics of cell growth and death, substrate consumption (glutamine and glucose) and metabolites production (lactate and ammonia). Based on a sensitivity analysis of the model outputs with respect to the parameters, a model reduction is proposed. Finally, the maximization of biomass productivity of hybridoma cell fed-batch cultures is analyzed. This model allows, on the one hand, quantitatively describing overflow metabolism in mammalian cell cultures and, on the other hand, will be valuable for monitoring and control of fed-batch cultures in order to optimize the process. This is illustrated in this contribution with the determination of optimal feeding profiles aiming at maximizing biomass productivity.     

Flow cytometry and cell cycle kinetics in continuous and fed-batch fermentations of budding yeast.

Cell size distributions, obtained either as protein distribution by flow cytometry or as cell volume distribution by a Coulter counter, give relevant information about the growth conditions of populations of budding yeast Saccharomyces cerevisiae. We have previously found a good correlation between these distributions and the growth rate in continuous cultures (Ranzi et al., Biotechnol. Bioeng. 1986, 28, 185-190). We now present determinations of the protein distributions and cell volume distributions during different fed-batch fermentations performed with a simple on/off controller. Since during the fed-batch fermentation a true steady state is not obtained, the distributions continuously change with time, but nevertheless we observed a good correlation between the average of both distributions and the actual growth rate. The behavior of the cell size distributions can be interpreted on the basis of a two-threshold cell cycle model in which both the critical protein content at budding (Ps) and the critical protein content for cell division (Pm) are differently modulated by the growth rate. Additional findings will be presented showing that this model can be used to successfully explain the insurgence and the maintenance of oscillatory states in continuous cultures.     

Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures. In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.     

Evaluation of mammalian fed-batch cultivations by two different models

Simulations of fed-batch hybridoma cell cultures were performed with two different models, a new model suggested by Nielsen and an older model suggested by Batt and Kompala. The response of the new Nielsen model was less sensitive to changes in feed concentrations and frequencies than the Batt and Kompala model. The new simulations with the Nielsen model as well as those previously with the Batt and Kompala model indicate, that possible benefits of fed-batch cultivations must be sought in other features than in intrinsic metabolic kinetics.     

Applications of improved stoichiometric model in medium design and fed-batch cultivation of animal cells in bioreactor

In our previous work (Xie and Wang, 1994a), a simplified stoichiometric model on energy metabolism for animal cell cultivation was developed. Fed-batch experiments were performed in T-flasks using this model in supplemental medium design (Xie and Wang, 1994b). In this work, the major pathways of glucose and glutamine metabolism were incorporated into the stoichiometric model. Fed-batch culture was conducted in a 2-liter bioreactor with appropriate process control strategies. Nutrient concentrations, especially glucose and glutamine, were maintained at constant but low levels through the automated feeding of a supplemental medium formulated using the improved stoichiometric model. The formation of toxic byproducts, such as ammonia and lactate (Hassellet al., 1991), was greatly reduced. The specific lactate production rate was decreased by 62-fold compared with batch culture in bioreactor and by 8-fold compared to fed-batch culture in T-flask using the previous stoichiometric model. Ammonia formation was also decreased compared with both the batch and fed-batch cultures. Most importantly, the monoclonal antibody concentration reached 900 mg l^?1, an increase of 17- and 1.6-fold compared with the batch and fed-batch cultures respectively.     

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%.     

A novel amino acid supplementation strategy based on a stoichiometric model to enhance human IL-2 (interleukin-2) expression in high-cell-density Escherichia coli cultures

A novel amino acid supplementation strategy was developed for enhancing the production of IL-2 (interleukin-2; as a model protein) by recombinant Escherichia coli BL21 (pET21a-hil2) in fed-batch high-cell-density cultures. The amino acids most needed and their amounts were determined using a stoichiometric model, and full factorial design experiments were conducted to determine the effects of single amino acids and amino acid mixtures on production. One of the most effective amino acid mixtures was found to be leucine, aspartic acid and glycine. This amino acid mixture was utilized for the production of IL-2 in batch and fed-batch fermentations. The amount of IL-2 produced increased from 403 to 722 mg/l and from 5.15 × 103 to 8.08 × 103 mg/l in batch and fed-batch cultures respectively. The results also revealed that the above amino acid mixture specifically increases IL-2 concentration in the cells.     

Use of chemostat data for modelling extracellular-inulinase production by Kluyveromyces marxianus in a high-cell-density fed-batch process

Production of extracellular inulinase by low-cell-density (2 kg dry weight·m⁻³) sucrose-limited chemostat cultures of Kluyveromyces marxianus obeyed saturated kinetics at dilution rates ranging from 0.02 to 0.5 h⁻¹. A non-structured Monod-type equation, describing the relation between specific growth rate and specific extracellular-inulinase production rate, was used to fit experimental data. THis equation was subsequently incorporated in a model for the production of biomass and extracellular inulinase in a high-cell-density (> 100 kg dry weight·m⁻³) fed-batchculture of K. marxianus grown on sucrose. The model adequately described biomass production in the fed-batch culture. However, the production of extracellular inulinase in the fed-batch process was slightly higher than predicted by the model. This observation may be related to differences in growth conditions between in the chemostat and fed-batch cultures.     

Energy metabolism and ATP balance in animal cell cultivation using a stoichiometrically based reaction network

A metabolic reaction network is developed for the estimation of the stoichiometric production of adenosine triphosphate (ATP) in animal cell culture. By using the material balance data from fed-batch and batch cultures of hybridoma cells, the stoichiometric ATP productions are determined with estimated effective P/O ratios of 2 for NADH and 1.2 for FADH(2). A significant percentage of the ATP requirement (16-41%) in hybridoma cells is generated directly from free energy release without the participation of oxygen. The oxidative phosphorylation of NADH accounts for about 60% of the total ATP production in the fed-batch cultures and about 47% in the batch culture. The oxidative phosphorylation of FADH(2) accounts for less then 20% of the total ATP production in all cases.A fractional model is devised to analyze the contribution of each nutrient to the ATP production. Results show that a majority of the ATP is produced from glucose metabolism (60-76%). Less than 30% of the ATP is derived from glutamine, and less than 11% is derived from other essential amino acids. The analysis also shows that the glycolytic pathway generates more ATP in the batch (41%) than in the fed-batch (<27%) cultures. The TCA cycle provides 51-68% of the total ATP production. The calculated stoichiometric oxygen consumption differs among the batch and fed-batch cultures, depending on the glucose concentration. This result suggests that the relationship between the oxygen uptake rate (OUR) and cell growth may change with the culture conditions. However, the calculated respiratory quotient (RQ) is relatively constant in all cases.A linear relationship is obtained between the specific ATP production rate and the specific cell growth rate. The maximum ATP yield and the maintenance ATP requirement are determined based on this linear relationship. The biosynthetic ATP demand estimated from the dry cell weight and cell composition is significantly lower than that calculated from the maximum ATP yield, indicating that the non-growth-associated ATP demand may contain other factors than what is considered in the estimation of the biosynthetic ATP demand. (c) 1996 John Wiley & Sons, Inc.     

Dynamic model of CHO cell metabolism

Fed-batch cultures are extensively used for the production of therapeutic proteins. However, process optimization is hampered by lack of quantitative models of mammalian cellular metabolism in these cultures. This paper presents a new kinetic model of CHO cell metabolism and a novel framework for simulating the dynamics of metabolic and biosynthetic pathways of these cells grown in fed-batch culture. The model defines a subset of the intracellular reactions with kinetic rate expressions based on extracellular metabolite concentrations and temperature- and redox-dependent regulatory variables. The simulation uses the rate expressions to calculate pseudo-steady state flux distributions and extracellular metabolite concentrations at discrete time points. Experimental data collected in this study for several different CHO cell fed-batch cultures are used to derive the rate expressions, fit the parameters, and validate the model. The simulations accurately predicted the effects of process variables, including temperature shift, seed density, specific productivity, and nutrient concentrations.     

Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110.

Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fed-batch culture of insect cells with exponential feeding of amino acid and yeastolate solution

Amino acids rather than sugars are the primary limiting substrates for the culture of insect cells in a Grace's medium. When cultures are supplemented with amino acids, the yeastolate components other than the amino acids become the secondary limiting substrates. For the fed-batch culture of insect cells, a solution containing concentrated amino acids and yeastolate was supplied using an exponential feed flow rate calculated from mass balance equations. During the batch period the specific growth rate was 0.02 h^у, whereas during the fed-batch period it was measured as 0.007 and 0.012 h^у on the basis of the cell numbers and the dry cell weight, respectively. This difference in the specific growth rates in the fed-batch period is caused by an increase in the cell size during this period. Furthermore, in fed-batch cultures, dissolved oxygen was found to be a limiting factor for high cell-density cultures.     

Revisiting Verhulst and Monod models: analysis of batch and fed-batch cultures

The paper re-evaluates Verhulst and Monod models. It has been claimed that standard logistic equation cannot describe the decline phase of mammalian cells in batch and fed-batch cultures and in some cases it fails to fit somatic growth data. In the present work Verhulst, population-based mechanistic growth model was revisited to describe successfully viable cell density (VCD) in exponential and decline phases of batch and fed-batch cultures of three different CHO cell lines. Verhulst model constants, K, carrying capacity (VCD/ml or μg/ml) and r, intrinsic growth factor (h⁻¹) have physical meaning and they are of biological significance. These two parameters together define the course of growth and productivity and therefore, they are valuable in optimisation of culture media, developing feeding strategies and selection of cell lines for productivity. The Verhulst growth model approach was extended to develop productivity models for batch and fed-batch cultures. All Verhulst models were validated against blind data (R² > 0.95). Critical examination of theoretical approaches concluded that Monod parameters have no physical meaning. Monod-hybrid (pseudo-mechanistic) batch models were validated against specific growth rates of respective bolus and continuous fed-batch cultures (R² ≈ 0.90). The reduced form of Monod-hybrid model C_L/(K_L + C_L) describes specific growth rate during metabolic shift (R² ≈ 0.95). Verhulst substrate-based growth models compared favourably with Monod-hybrid models. Thus, experimental evidence implies that the constants in the Monod-hybrid model may not have physical meaning but they behave similarly to the biological constants in Michaelis–Menten enzyme kinetics, the basis of the Monod growth model.