Modeling of <Emphasis Type="Italic">Fusarium redolens</Emphasis> Dzf2 mycelial growth kinetics and optimal fed-batch fermentation for beauvericin production

Beauvericin (BEA) is a cyclic hexadepsipeptide mycotoxin with notable phytotoxic and insecticidal activities. Fusarium redolens Dzf2 is a highly BEA-producing fungus isolated from a medicinal plant. The aim of the current study was to develop a simple and valid kinetic model for F. redolens Dzf2 mycelial growth and the optimal fed-batch operation for efficient BEA production. A modified Monod model with substrate (glucose) and product (BEA) inhibition was constructed based on the culture characteristics of F. redolens Dzf2 mycelia in a liquid medium. Model parameters were derived by simulation of the experimental data from batch culture. The model fitted closely with the experimental data over 20–50 g l⁻¹ glucose concentration range in batch fermentation. The kinetic model together with the stoichiometric relationships for biomass, substrate and product was applied to predict the optimal feeding scheme for fed-batch fermentation, leading to 54% higher BEA yield (299 mg l⁻¹) than in the batch culture (194 mg l⁻¹). The modified Monod model incorporating substrate and product inhibition was proven adequate for describing the growth kinetics of F. redolens Dzf2 mycelial culture at suitable but not excessive initial glucose levels in batch and fed-batch cultures.     

Computer programs for modeling mammalian cell batch and fed-batch cultures using logistic equations

A MATLAB^® toolbox was developed for applying the logistic modeling approach to mammalian cell batch and fed-batch cultures. The programs in the toolbox encompass sensitivity analyses and simulations of the logistic equations in addition to cell specific rate estimation. The toolbox was first used to generate time courses of the sensitivity equations for characterizing the relationship between the logistic variable and the model parameters. Subsequently, the toolbox was used to describe CHO cell data from batch and fed-batch mammalian cell cultures. Cell density, product, glucose, lactate, glutamine, and ammonia data were analyzed for the batch culture while fed-batch analysis included cell density and product concentration. In all instances, experimental data were well described by the logistic equations and the resulting specific rate profiles were representative of the underlying cell physiology. The 6-variable batch culture data set was also used to compare the logistic specific rates with those from polynomial fitting and discrete derivative methods. The polynomial specific rates grossly misrepresented cell behavior in the initial and final stages of culture while those based on discrete derivatives had high variability due to computational artifacts. The utility of logistic specific rates to guide process development activities was demonstrated using specific protein productivity versus growth rate trajectories for the 3 cultures examined in this study. Overall, the computer programs developed in this study enable rapid and robust analysis of data from mammalian cell batch and fed-batch cultures which can help process development and metabolic flux estimation.     

Kinetic models for astaxanthin production by high cell density mixotrophic culture of the microalga Haematococcus pluvialis

High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light intensity control mode. A high cell density of 2.65 g L⁻¹ (batch culture) or 2.74 g L⁻¹ (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg L⁻¹) was about 20.5% higher than in the batch culture (53.43 mg L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as well as sodium acetate consumption was proposed. Good agreement was found between the model predictions and experimental data. The models demonstrated that the optimal light intensity for mixotrophic growth of H. pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90–360 μmol m⁻² s⁻¹, and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode. Received 24 December 1998/ Accepted in revised form 23 April 1999     

Kinetic models for phycocyanin production by high cell density mixotrophic culture of the microalga Spirulina platensis

Phycocyanin production by high cell density cultivation of Spirulina platensis in batch and fed-batch modes in 3.7-L bioreactors with a programmed stepwise increase in light intensity program was investigated. The results showed that the cell density in fed-batch culture (10.2 g L⁻¹) was 4.29-fold that in batch culture (2.38 g L⁻¹), and the total phycocyanin production in the fed-batch culture (0.795 g L⁻¹) was 3.05-fold that in the batch culture (0.261 g L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, phycocyanin formation, as well as glucose consumption was proposed. The data fitted the models well (r ² > 0.99). Furthermore, based on the kinetic models, the potential effects of light limitation and photoinhibition on cell growth and phycocyanin formation can be examined in depth. The models demonstrated that the optimal light intensity for mixotrophic growth of Spirulina platensis in batch or fed-batch cultures using a 3.7-L bioreactor was 80160 μE m⁻² s⁻¹, and the stepwise increase in light intensity can be replaced by a constant light intensity mode. Received 28 July 1998/ Accepted in revised form 8 October 1998     

Effect of Growth Conditions and Trehalose Content on Cryotolerance of Bakers' Yeast in Frozen Doughs

The cryotolerance in frozen doughs and in water suspensions of bakers' yeast (Saccharomyces cerevisiae) previously grown under various industrial conditions was evaluated on a laboratory scale. Fed-batch cultures were very superior to batch cultures, and strong aeration enhanced cryoresistance in both cases for freezing rates of 1 to 56°C min⁻¹. Loss of cell viability in frozen dough or water was related to the duration of the dissolved-oxygen deficit during fed-batch growth. Strongly aerobic fed-batch cultures grown at a reduced average specific rate (μ = 0.088 h⁻¹ compared with 0.117 h⁻¹) also showed greater trehalose synthesis and improved frozen-dough stability. Insufficient aeration (dissolved-oxygen deficit) and lower growth temperature (20°C instead of 30°C) decreased both fed-batch-grown yeast cryoresistance and trehalose content. Although trehalose had a cryoprotective effect in S. cerevisiae, its effect was neutralized by even a momentary lack of excess dissolved oxygen in the fed-batch growth medium.     

Kinetic and stoichiometric characterization of organoautotrophic growth of Ralstonia eutropha on formic acid in fed‐batch and continuous cultures

Formic acid, acting as both carbon and energy source, is a safe alternative to a carbon dioxide, hydrogen and dioxygen mix for studying the conversion of carbon through the Calvin–Benson–Bassham (CBB) cycle into value-added chemical compounds by non-photosynthetic microorganisms. In this work, organoautotrophic growth of Ralstonia eutropha on formic acid was studied using an approach combining stoichiometric modeling and controlled cultures in bioreactors. A strain deleted of its polyhydroxyalkanoate production pathway was used in order to carry out a physiological characterization. The maximal growth yield was determined at 0.16 Cmole Cmole⁻¹ in a formate-limited continuous culture. The measured yield corresponded to 76% to 85% of the theoretical yield (later confirmed in pH-controlled fed-batch cultures). The stoichiometric study highlighted the imbalance between carbon and energy provided by formic acid and explained the low growth yields measured. Fed-batch cultures were also used to determine the maximum specific growth rate (μ_max = 0.18 h⁻¹) and to study the impact of increasing formic acid concentrations on growth yields. High formic acid sensitivity was found in R eutropha since a linear decrease in the biomass yield with increasing residual formic acid concentrations was observed between 0 and 1.5 g l⁻¹.     

Formulation of a protein-free medium based on IPL-41 for the sustained growth of Drosophila melanogaster S2 cells

An animal protein-free medium was developed for Drosophila melanogaster S2 (S2AcGPV2) cells genetically modified to produce the rabies virus G glycoprotein (GPV). IPL-41, used as a basal medium, was supplemented with yeastolate, carbohydrates, amino acids and lipids aiming initially to reduce and further to eliminate the need of fetal bovine serum. The S2AcGPV2 cells were fully capable of growing in serum-free supplemented IPL-41 medium containing 6 g L⁻¹ yeastolate ultrafiltrate, 10 g L⁻¹ glucose, 3.5 g L⁻¹ glutamine, 0.5 g L⁻¹ fructose, 2 g L⁻¹ lactose, 0.6 g L⁻¹ tyrosine, 1.48 g L⁻¹ methionine and 1% (v/v) lipid emulsion, reaching 19 × 10⁶ cells mL⁻¹. Maximum specific growth rate and cell productivity were 0.025 h⁻¹ and 0.57 × 10⁵ cells mL⁻¹ h⁻¹, respectively. Glucose and lactose were consumed during cell culture, but not fructose. Lactate concentration generally decreased during cell culture, while ammonium concentration reached 167 mg L⁻¹, however, without noticeable deleterious effects on cell growth. GPV concentration values achieved were, however, modest in the proposed medium formulation.     

The effect of degree and timing of nitrogen limitation on lipid productivity in Chlorella vulgaris

Improvements in lipid productivity would enhance the economic feasibility of microalgal biodiesel. In order to optimise lipid productivity, both the growth rate and lipid content of algal cells must be maximised. The lipid content of many microalgae can be enhanced through nitrogen limitation, but at the expense of biomass productivity. This suggests that a two-stage nitrogen supply strategy might improve lipid productivity. Two different nitrogen supply strategies were investigated for their effect on lipid productivity in Chlorella vulgaris. The first was an initial nitrogen-replete stage, designed to optimise biomass productivity, followed by nitrogen limitation to enhance lipid content (two-stage batch) and the second was an initial nitrogen-limited stage, designed to maximise lipid content, followed by addition of nitrogen to enhance biomass concentration (fed-batch). Volumetric lipid yield in nitrogen-limited two-stage batch and fed-batch was compared with that achieved in nitrogen-replete and nitrogen-limited batch culture. In a previous work, maximum lipid productivity in batch culture was found at an intermediate level of nitrogen limitation (starting nitrate concentration of 170 mg L^?1). Overall lipid productivity was not improved by using fed-batch or two-stage culture strategies, although these strategies showed higher volumetric lipid concentrations than nitrogen-replete batch culture. The dilution of cultures prior to nitrogen deprivation led to increased lipid accumulation, indicating that the availability of light influenced the rate of lipid accumulation. However, dilution did not lead to increased lipid productivity due to the resulting lower biomass concentration.     

Comparison of polysialic acid production in Escherichia coli K1 during batch cultivation and fed-batch cultivation applying two different control strategies

The polysialic acid (PSA) production in Escherichia coli (E. coli) K1 was studied using three different cultivation strategies. A batch cultivation, a fed-batch cultivation at a constant specific growth rate of 0.25 h⁻¹ and a fed-batch cultivation at a constant glucose concentration of 50 mg l⁻¹ was performed. PSA formation kinetics under different cultivation strategies were analyzed based on the Monod growth model and the Luedeking-Piret equation. The results revealed that PSA formation in E. coli K1 was completely growth associated, the highest specific PSA formation rate (0.0489 g g⁻¹ h⁻¹) was obtained in the batch cultivation. However, comparing biomass and PSA yields on the glucose consumed, both fed-batch cultivations provided higher yields than that of the batch cultivation and acetate formation was prevented. Moreover, PSA yield on glucose was also correlated to the specific growth rate of the cells. The optimal specific growth rate for PSA production was 0.32 h⁻¹ obtained in the fed-batch cultivation at a constant glucose concentration of 50 mg l⁻¹, with highest conversion efficiency of 43 mg g⁻¹.     

Cultivation of Chlorella pyrenoidosa in soybean processing wastewater

Chlorella pyrenoidosa was cultivated in soybean processing wastewater (SPW) in batch and fed-batch cultures without a supply of additional nutrients. The alga was able to remove 77.8 ± 5.7%, 88.8 ± 1.0%, 89.1 ± 0.6% and 70.3 ± 11.4% of soluble chemical oxygen demand (SCOD_Cr), total nitrogen (TN), NH₄⁺-N and total phosphate (TP), respectively, after 120 h in fed-batch culture. C. pyrenoidosa attained an average biomass productivity of 0.64 g L⁻¹ d⁻¹, an average lipid content of 37.00 ± 9.34%, and a high lipid productivity of 0.40 g L⁻¹ d⁻¹. Therefore, cultivation of C. pyrenoidosa in SPW could yield cleaner water and useful biomass.     

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.     

Fed-batch culture optimization of a growth-associated hybridoma cell line in chemically defined protein-free media

An investigation was made to study the processes of fed-batch cultures of a hybridoma cell line in chemically defined protein-free media. First of all, a strong growth-associated pattern was correlated between the production of MAb and growth of cells through the kinetic studies of batch cultures, suggesting the potential effectiveness of extending the duration of exponential growth in the improvement of MAb titers. Second, compositions of amino acids in the feeding solution were balanced stepwisely according to their stoichiometrical correlations with glucose uptake in batch and fed-batch cultures. Moreover, a limiting factor screening revealed the constitutive nature of Ca²⁺ and Mg²⁺ for cell growth, and the importance of their feeding in fed-batch cultures. Finally, a fed-batch process was executed with a glucose uptake coupled feeding of balanced amino acids together with groups of nutrients and a feeding of CaCl₂ and MgCl₂ concentrate. The duration of exponential cell growth was extended from 70 h in batch culture and 98 h in fed-batch culture without Ca²⁺/Mg²⁺ feeding to 117 h with Ca²⁺/Mg²⁺ feeding. As a result of the prolonged exponential cell growth, the viable and total cell densities reached 7.04 × 10⁶ and 9.12 × 10⁶ cells ml⁻¹, respectively. The maximal MAb concentration achieved was increased to approximately eight times of that in serum supplemented batch culture.     

Evaluation of high salinity adaptation for lipid bio-accumulation in the green microalga Chlorella vulgaris

Aiming at the reutilizing wastewater for algal growth and biomass production, a saline water rejected from reverse osmosis (RO) facility (salinity 67.59 g L⁻¹) was used to cultivate the pre-adapted green microalga Chlorella vulgaris. The inoculum was prepared by growing cells in modified BG-11 medium, and adaptation was performed by applying a gradual increase in salinity (56.0 g L⁻¹ NaCl and 125 ppm FeSO₄·7H₂O) to the culture in 200 L photobioreactor. Experiments using the adapted alga were performed using original-rejected water (ORW) and treated rejected water (TRW) comparing with the recommended growth medium (BG-11). The initial salinity of ORW was chemically reduced to 39.1 g L⁻¹ to obtain TRW. Vertical photobioreactors (15 L) was used for indoor growth experiments. Growth in BG-11 resulted in 1.23 g L⁻¹, while the next adaptation growth reached 2.14 g L⁻¹ of dry biomass. The dry weights of re-cultivated Chlorella after adaptation were 1.49 and 2.19 g L⁻¹ from ORW and TRW; respectively. The cellular oil content was only 12% when cells grown under control conditions verses to 14.3 and 15.42% with original and treated water, respectively. Induction of stress affected the fatty acid methyl esters (FAMEs) profile and the properties of the resulting biodiesel. The present results indicated that induction of stress by high salinity improves the quality of FAMEs that can be used as a promising biodiesel fuel.     

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

Many studies underlined the great benefits of hydrolysates used as additives in animal free media on cell culture performances. However, to precisely define hydrolysate supplementation strategies, a deeper understanding of their effect on cell growth and protein production is required. In the present study, the effect of addition of one yeast extract (YE) and two yeast peptones (named YP.A and YP.B) in a chemically defined medium was first assessed on cell culture performances. Interestingly, specific effects were found depending on the degree of degradation of yeast hydrolysates. The YE at 1 g L⁻¹ increased the maximal cell density by 70 %, while a mixture of YE (1 g L⁻¹) and YP.A (4 g L⁻¹) increased IgG production by 180 %. These conditions were then evaluated on the CHO cell kinetics all over cultures. Hydrolysates extended the cell growth phase in Erlenmeyer flask and increased the maximal growth rate in bioreactor up to 20 %. Cell growth stimulation induced by hydrolysates addition was linked with energetic metabolism improvement suggesting that they promote oxidative pathway. Furthermore, hydrolysates provided an additional source of substrate that supported cell growth despite glutamine limitation.     

Effects of clonal variation on growth, metabolism, and productivity in response to trophic factor stimulation: a study of Chinese hamster ovary cells producing a recombinant monoclonal antibody

The growth, metabolism, and productivity of five Chinese hamster ovary (CHO) clones were explored in response to stimulation with insulin (5 mg/L) and LONG^®R³IGF-I (20 μg/L or 100 μg/L). All five clones were derived from the same parental CHO cell line (DG44) and produced the same recombinant monoclonal antibody, with varying specific productivities. There was no uniform response among the clones to stimulation with the different trophic factors. One of the high productivity clones (clone D) exhibited significantly better growth in response to LONG^®R³IGF-I; whereas the other clones showed equivalent or slightly better growth in the presence of insulin. Three out of the five clones had higher specific productivities in the presence of insulin (although not statistically significant); one was invariant, and the final clone exhibited slightly higher specific productivity in the presence of LONG^®R³IGF-I. Total product titers exhibited moderate variation between culture conditions, again with neither trophic factor being clearly superior. Overall product titers were affected by variations in both integrated viable cell density and specific productivity. Nutrient uptake and metabolite generation patterns varied strongly between clones and much less with culture conditions. These results point to the need for careful clonal analysis when selecting clones, particularly for platform processes where media and culture conditions are predetermined.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-011-9388-z) contains supplementary material, which is available to authorized users.     

A new strategy for fed-batch process control of HEK293 cell cultures based on alkali buffer addition monitoring: comparison with O.U.R. dynamic method

The increasing demand for biopharmaceuticals produced in mammalian cells has driven the industry to enhance productivity of bioprocesses through different strategies. This is why fed-batch and perfusion cultures are considered more attractive choices than batch processes. In this context, the availability of reliable online measuring systems for cell density and metabolic activity estimation will help the application of these processes. The present work focuses on the comparison of two different monitoring tools for indirect estimation of biomass concentration in a HEK293 cell cultures producing IFN-γ: on one side, the oxygen uptake rate (O.U.R.) determination, by means of application of the dynamic method measurement which is already a widely used tool and, on the other side, a new robust online monitoring tool based on the alkali buffer addition used to maintain the pH set point. Both strategies allow a proper monitoring of cell growth and metabolic activity, with precise identification of the balanced cell growth and the most important action in the process, as is the media feeding. The application of these monitoring systems in fed-batch processes allows extending the growth of HEK293 cells, which in turn results in higher final cell concentrations compared with Batch strategy (7 · 10⁶ cells mL⁻¹), achieving 14 · 10⁶ cells mL⁻¹ for the fed-batch based on O.U.R. and 19 · 10⁶ cells mL⁻¹ for the fed-batch based on the alkali addition. Product titter is also increased in respect of the batch strategy (3.70 mg L⁻¹), resulting in 8.27 mg L⁻¹ when fed-batch was based on O.U.R. and 11.49 mg L⁻¹ when it was based on the alkali buffer strategy. Results prove that fed-batch strategy based on the alkali buffer addition is a robust online monitoring method that has shown its great potential to optimize the feeding strategy in HEK293 fed-batch cultures.

     

Kinetic study of Lactococcus lactis strains (SLT6 and SLT10) growth on papain-hydrolysed whey

The effect of controlled whey hydrolysis by papain on growth of two lactic acid bacteria isolated from artisanal Leben: Lactococcus lactis var. diacetylactis (SLT6 and SLT10) was investigated. The higher biomass and maximum specific growth rate (μ _max) were obtained after 30 min of hydrolysis. HPLC analysis of peptides showed that whey hydrolysis reduced the amount of peptides of MW > 400 Da and increased those peptides of MW < 400 Da. The two studied strains exhibited different peptide requirements. The pH-controlled batch cultures in 30 min hydrolysed whey followed the Monod kinetic for growth and for lactate production. The values of the key kinetic constants were: maximum specific growth rate (μ _max), 1.08 and 0.56 h^?1; yield biomass on lactose (Y _x/s), 0.20 and 0.18 g g^?1 and saturation constant K _s, 4.2 and 2.8 g L^?1 for SLT6 and SLT10, respectively. When compared with batch experimental data, the model provided good predictions for growth, lactose utilisation and lactate production profiles.     

Effects of carbon sources on growth and extracellular polysaccharide production of Nostoc flagelliforme under heterotrophic high-cell-density fed-batch cultures

Dissociated cells separated from a natural colony of Nostoc flagelliforme were cultivated heterotrophically in the darkness on glucose under fed-batch culture conditions. The effects of carbon sources (glucose, fructose, xylose, and sucrose) and concentrations on cell growth and extracellular polysaccharide (EPS) production were investigated. At harvest, the culture contained 1.325 g L^?1 of biomass and 117.2 mg L^?1 of EPS, respectively. The gravimetric EPS production rate was 16.7 mg g^?1 cell dry weight day^?1, which was 2.1 times higher than previously reported. Using sigmoid model, batch fermentation of N. flagelliforme was kinetically simulated to obtain equations including substrate consumption, biomass growth, and EPS accumulation. Results from a simulation correlated well with the experimental ones, indicating that this method could be useful in studying EPS production from batch and fed-batch cultures.     

Cytotoxicity and genotoxicity of zingiberene on different neuron cell lines in vitro

The main objective of this study is to investigate the cytotoxic, genotoxic and antioxidant properties of zingiberene (ZBN) in an in vitro rat brain cell culture study. The cytotoxic effect was determined against the rat neuron and N2a neuroblastoma (N2a-NB) cell lines using the 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while the antioxidant activity was assessed using the total antioxidant capacity (TAC) and total oxidative stress (TOS) assays. The effects on DNA damage were also evaluated in this study by the single cell gel electrophoresis assay. The results indicated that ZBN has an anti-proliferative activity suppressing the proliferation of N2a-NB cells at concentrations over 50 mg L⁻¹ and neuron cells at concentrations over 150 mg L⁻¹. In addition, ZBN treatments at higher doses (≤50 mg L⁻¹) led to increases of TOS levels in N2a-NB cell cultures. However 25 mg L⁻¹ of ZBN treatment caused increases of TAC levels in cultured neuron and N2a-NB cell cultures while ZBN at doses of 10–400 mg L⁻¹ did not increase the number of total damage score in both cell lines. This study clearly indicates that ZBN has a significant potential to be used as a natural anticancer agent in cultured N2a-NBs.     

Ethanol effect on batch and fed-batch Arthrospira platensis growth

The ability of Arthrospira platensis to use ethanol as a carbon and energy source was investigated by batch process and fed-batch process. A. platensis was cultivated under the effect of a single addition (batch process) and a daily pulse feeding (fed-batch process) of pure ethanol, at different concentrations, to evaluate cell concentration (X) and specific growth rate (μ). A marked increase was observed in the cell concentration of A. platensis in runs with ethanol addition when compared to control cultures without ethanol addition. The fed-batch process using an ethanol concentration of 38 mg L^?1 days^?1 reached the maximum cell concentration of 2,393 ± 241 mg L^?1, about 1.5-fold that obtained in the control culture. In all experiments, the maximum specific growth rate was observed in the early exponential phase of cell growth. In the fed-batch process, μ decreased more slowly than in the batch process and control culture, resulting in the highest final cell concentration. Ethanol can be used as a feasible carbon and energy source for A. platensis growth via a fed-batch process.