Bioreactor operating strategy inThalictrum rugosum plant cell culture for the production of berberine

Optimal substrate feeding strategy in bioreactor operation was investigated to increase the production of secondary metabolite in a high density culture of plant cell. It was accomplished by the previously proposed structured kinetic model that describes the cell growth and synthesis of the secondary metabolite, berberine, in a batch suspension culture ofThalictrum rugosum. Four types of operation strategies for sugar feeding intoT. rugosum culture were proposed based on the model, which were the periodic fedbatch operations to maintain the cell activity, the cell viability, and the specific production rate, and the perfusion operation to maintain the specific production rate. From the simulation results of these strategies, it could be found that the periodic fed-batch operation and the perfusion operation could achieve the higher volumetric production of berberine (mg berberine/L) and specific production yield (mg berberine/g dry cell weight) than those of batch cultures. Although the highest productivity (mg berberine/day) of berberine could be achieved by the periodic fed-batch operation to maintain the cell activity compared with the other strategies in the periodic fed-batch operations, the specific production yield was low due to the higher maximum dry cell weight than other cases. The periodic fed-batch operation to maintain cell viability resulted in the highest volumetric production of berberine and specific production yield compared with the other strategies. In the cases of maintaining the specific production rate, the per-formance of the periodic fed-batch operation was better than that of the perfusion operation in the respect of the volumetric production and productivity of berberine. In order to increase the volumetric production of berberine and to get the highest specific production yield, the periodic fed-batch operation to maintain cell viability could be chosen as the optimal operating strategy in high density, culture ofT. rugosum plant cell.     

Enhanced hyaluronic acid production by a two-stage culture strategy based on the modeling of batch and fed-batch cultivation of Streptococcus zooepidemicus

This study aimed to enhance hyaluronic acid (HA) production by a two-stage culture strategy based on the modeling of batch and fed-batch culture of Streptococcus zooepidemicus. Batch culture had higher specific HA synthesis rate while fed-batch culture had higher specific cell growth rate. The lower specific HA synthesis rate in fed-batch culture resulted from the competition of cell growth for the common precursors at a low substrate concentration. Based on the modeling of batch and fed-batch culture of S. zooepidemicus, a two-stage culture strategy was proposed to enhance HA production. S. zooepidemicus were cultured in a fed-batch mode with sucrose concentration maintained at 1.0+/-0.2g/L during 0-8h and then batch culture was performed during 8-20h with an initial sucrose concentration of 15g/L. With the proposed two-stage culture strategy, HA production was increased to 6.6g/L compared with 5.0g/L in batch culture with the same total sucrose. The enhanced HA production by the proposed two-stage culture strategy resulted from the decreased inhibition of cell growth and the increased transformation rate of sucrose to HA.     

Process development for an inducible rituximab-expressing Chinese hamster ovary cell line

Inducible mammalian expression systems are becoming increasingly available and are not only useful for the production of cytotoxic/cytostatic products, but also confer the unique ability to uncouple the growth and production phases. In this work, we have specifically investigated how the cell culture state at the time of induction influences the cumate-inducible expression of recombinant rituximab by a GS-CHO cell line. To this end, cells grown in batch and fed-batch cultures were induced at increasing cell densities (1 to 10 × 10 ⁶ cells/mL). In batch, the cell specific productivity and the product yield were found to reduce with increasing cell density at induction. A dynamic feeding strategy using a concentrated nutrient solution applied prior and postinduction allowed to significantly increase the integral of viable cells and led to a 3-fold increase in the volumetric productivity (1.2 g/L). The highest product yields were achieved for intermediate cell densities at induction, as cultures induced during the late exponential phase (10 × 10 ⁶ cells/mL) were associated with a shortened production phase. The final glycosylation patterns remained however similar, irrespective of the cell density at induction. The kinetics of growth and production in a 2 L bioreactor were largely comparable to shake flasks for a similar cell density at induction. The degree of galactosylation was found to decrease over time, but the final glycan distribution at harvest was consistent to that of the shake flasks cultures. Taken together, our results provide useful insights for the rational development of fed-batch cell culture processes involving inducible CHO cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2742, 2019     

Fed-batch culture of<Emphasis Type="Italic"> Bacillus thuringiensis</Emphasis> based on motile intensity

The operation of a fed-batch culture is more complicated than that of batch or continuous culture. Thus, an appropriate feeding strategy for fed-batch cultures should be carefully designed. In this study, a simple feeding strategy for fed-batch culture of Bacillus thuringiensis based on motile intensity is described. The feeding strategy consisted of two steps: (1) initiating feeding at the peak of motile intensity; (2) terminating feeding at low motile intensity (or non-motility) of the cells. In addition, the motile intensity of B. thuringiensis was used to determine the optimum environmental conditions (pH, temperature, and dissolved oxygen) and optimum medium composition. Using this fed-batch strategy, the production of thuringiensin increased 34% compared with batch culture using the same environmental conditions and medium composition. The proposed strategy for fed-batch culture helps to avoid overfeeding of substrate and facilitates on-line control. A comparison of several alternative strategies for fed-batch culture demonstrated that strategies such as glucose-stat and DO-stat result in a lower productivity than that obtained using the motility intensity method.     

Enhanced 2,3-butanediol production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> SDM

Enhanced 2,3-butanediol (BD) production was carried out by Klebsiella pneumoniae SDM. The nutritional requirements for BD production by K. pneumoniae SDM were optimized statistically in shake flask fermentations. Corn steep liquor powder and (NH₄)₂HPO₄ were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform fed-batch fermentations with K. pneumoniae SDM. BD production was then studied in a 5-l bioreactor applying different fed-batch strategies, including pulse fed batch, constant feed rate fed batch, constant residual glucose concentration fed batch, and exponential fed batch. The maximum BD concentration of 150 g/l at 38 h with a diol productivity of 4.21 g/l h was obtained by the constant residual glucose concentration feeding strategy. To the best of our knowledge, these results were new records on BD fermentation. Cuiqing Ma and Ailong Wang contributed equally to this work.     

Growth and carotenoid production of Phaffia Rhodozyma in fed-batch cultures with different feeding methods

The growth and carotenoid production of Phaffia rhodozyma in fed-batch cultures with different feeding methods and grown at specific growth rates similar to the batch culture was compared. With constant feeding, exponential feeding, DO-stat and pH-stat fed-batch cultures of Phaffia rhodozyma, the highest biomass (17.4 g/l) and lowest carotenoid content (307 g/g cell) of Phaffia rhodozyma was from the DO-stat fed-batch culture. The lowest biomass (14.7 g/l) and highest carotenoid content (412 g/g cell) was from the exponential, fed-batch culture.     

Fed-batch fermentation for production of nitrile hydratase byRhodococcus rhodochrous M33

To enhance the productivity and activity of nitrile hydratase inRhodococcus rhodochrous M33, a glucose-limited fed-batch culture was performed. In a fed-batch culture where the glucose was controlled at a limited level and cobalt was supplemented during the fermentation period, the cell mass and total activity of nitrile hydratase both increased 3.3-fold compared to that in the batch fermentation. The productivity of nitrile hydratase also increased 1.9-fold compared to that in the batch fermentation. The specific activity of nitrile hydratase in the whole cell preparation when using a fed-batch culture was 120 units/mg-DCW, which was similar to that in the batch culture.     

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.     

Analysis of metabolic fluxes for hyaluronic acid (HA) production by Streptococcus zooepidemicus

Summary A detailed metabolic flux analysis (MFA) for hyaluronic acid (HA) production by Streptococcus zooepidemicus was carried out. A metabolic network was constructed for the metabolism of S. zooepidemicus. Fluxes through these reactions were estimated by MFA using accumulation rates of biomass and product, consumption rate of glucose in batch fermentation and dissolved oxygen-controlled fermentation. The changes of the fluxes were observed at different stages of batch fermentation and in different dissolved oxygen tension (DOT)-controlled fermentation processes. The effects of metabolic nodes on HA accumulation under various culture conditions were investigated. The results showed that high concentration of glucose in the medium did not affect metabolic flux distribution, but did influence the uptake rate of glucose. HA synthesis was influenced by DOT via flux redistribution in the principal node. Adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) produced in the fermentation process are associated with cell growth and HA synthesis.     

Effect of growth rate on the production of<Emphasis Type="SmallCaps">l</Emphasis>-proline in the fed-batch culture of<Emphasis Type="Italic">Corynebacterium acetoacidophilum</Emphasis>

Corynebacterium acetoacidophilum RYU3161 was cultivated in al-histidine-limited fed-batch culture. To investigate the effect of cell growth on thel-proline production, 5l fed-batch culture was performed using an exponential feeding rate to obtain the specific growth rates (μ) of 0.04, 0.06, 0.08, and 0.1 h⁻¹. The results show that the highest production ofl-proline was obtained at μ=0.04 h⁻¹. The specificl-proline production rate (Q_p) increased proportionally as a function of the specific growth rate, but decreased after it revealed the maximum value at μ=0.08 h⁻¹. Thus, the highest productivity ofl-proline was 1.66 g L⁻¹ h⁻¹ at μ=0.08 h⁻¹. The results show that the production of L-proline inC. acetoacidophilum RYU3161 has mixed growth-associated characteristics.     

Overproduction of glutathione by <Emphasis Type="SmallCaps">l</Emphasis>-cysteine addition and a temperature-shift strategy

The present study investigated the effects of three constituent amino acids on glutathione production in flask culture of Candida utilis. Although l-glutamic acid and glycine had little impact on cell growth and glutathione biosynthesis, l-cysteine positively influenced glutathione production, despite inhibiting cell growth when it was added prior to stationary phase. Adding 8 mmol/L of l-cysteine to the culture broth at 16 h boosted glutathione production by 91%, increasing the intracellular glutathione content by 106% compared to untreated controls. A temperature-shift strategy, in which we shifted batch and fed-batch cultures of C. utilis from 30 to 26°C, also significantly enhanced glutathione production. Applying both strategies (i.e. adding 20 mmol/L l-cysteine and shifting the temperature from 30 to 26°C) at 33 h enhanced the glutathione concentration and the intracellular glutathione content to 1,312 mg/L and 3.75%, respectively, during fed-batch cultivation (glucose feeding at a constant rate of 18.3 g/h). The average specific glutathione production rate under this condition was 129% higher than that of the control without strategy.     

High cell density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> with surface anchored transglucosidase for use as whole-cell biocatalyst for α-arbutin synthesis

A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for α-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of α-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.     

Multi-stage high cell continuous fermentation for high productivity and titer

We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody.     

Poly(3-Hydroxybutyrate) Production with High Productivity and High Polymer Content by a Fed-Batch Culture of Alcaligenes latus under Nitrogen Limitation

Alcaligenes latus has been known to produce poly(3-hydroxybutyrate) (PHB) in a growth-associated manner even under nutrient-sufficient conditions. However, the PHB content obtained by fed-batch culture was always low, at ca. 50%, which makes the recovery process inefficient. In this study, the effect of applying nitrogen limitation on the production of PHB by A. latus was examined. In flask and batch cultures, the PHB synthesis rate could be increased considerably by applying nitrogen limitation. The PHB content could be increased to 87% by applying nitrogen limitation in batch culture, which was considerably higher than that typically obtainable (50%) under nitrogen-sufficient conditions. In fed-batch culture, cells were first cultured by the DO-stat feeding strategy without applying nitrogen limitation. Nitrogen limitation was applied at a cell concentration of 76 g (dry cell weight)/liter, and the sucrose concentration was maintained within 5 to 20 g/liter. After 8 h of nitrogen limitation, the cell concentration, PHB concentration, and PHB content reached 111.7 g (dry cell weight)/liter, 98.7 g/liter, and 88%, respectively, resulting in a productivity of 4.94 g of PHB/liter/h. The highest PHB productivity, 5.13 g/liter/h, was obtained after 16 h.     

Exploring low-cost carbon sources for microbial lipids production by fed-batch cultivation of <Emphasis Type="Italic">Cryptococcus albidus</Emphasis>

Volatile fatty acids (VFAs), acetic acid, acetates, and ethanol were used as carbon sources for the production of microbial lipids using Cryptococcus albidus in batch cultures. C. albidus utilized organic acids less than glucose in the production of lipids, resulting in a lipid yield coefficient on VFAs of 0.125 g/g. In a two-stage batch culture, the lipid content increased to 43.8% (w/w) when VFAs were used as the sole carbon source in the second stage, which was two times higher than that of the batch culture. Furthermore, a 192 h, two-stage fed-batch cultivation of C. albidus produced a dry cell weight, lipid concentration, and lipid content of 26.4 g/L, 14.5 g/L, and 55.1% (w/w), respectively. The fed-batch culture model used in this study featured pure VFA solutions, with intermittent feeding, under oxygen-enriched air supply conditions. This study investigated several alternative carbon sources to reduce the cost of microbial lipids production and proved the feasibility of using VFAs as the carbon source for the provision of a high lipid content and productivity.     

Production of β-carotene by recombinant Escherichia coli with engineered whole mevalonate pathway in batch and fed-batch cultures

Recombinant Escherichia coli engineered to contain the whole mevalonate pathway and foreign genes for β-carotene biosynthesis, was utilized for production of β-carotene in bioreactor cultures. Optimum culture conditions were established in batch and pH-stat fed-batch cultures to determine the optimal feeding strategy thereby improving production yield. The specific growth rate and volumetric productivity in batch cultures at 37°C were 1.7-fold and 2-fold higher, respectively, than those at 28°C. Glycerol was superior to glucose as a carbon source. Maximum β-carotene production (titer of 663 mg/L and overall volumetric productivity of 24.6 mg/L × h) resulted from the simultaneous addition of 500 g/L glycerol and 50 g/L yeast extract in pH-stat fed-batch culture.     

A comparison of different culture methods for hybridoma propagation and monoclonal antibody production

A major variable to consider in the production of biologicals from mammalian cell cultures is the mode of operation, be it a batch, continuous, perfusion, fed-batch or other production method. The final choice must consider a number of fundamental and economic issues. Here we present some antibody production data from different cell lines using different modes of production and discuss the important factors for consideration in choosing a production strategy. It was found that the productivity of batch cultures was lower than that obtained in continuous and perfused cultures, but that productivity could be improved by implementing suitable feeding strategies. The antibody productivity of one cell line, MCL1, during exponential phase was not affected by media type or glucose level. The maximum productivity of two cell lines in continuous culture was found to occur at dilution rates below the maximum, from 0.019 to 0.030 hr^–1.     

Indigestible dextrin stimulates glucoamylase production in submerged culture of <Emphasis Type="Italic">Aspergillus kawachii</Emphasis>

Submerged batch cultures of Aspergillus kawachii grown on indigestible dextrin were investigated for potential improvements in glucoamylase (GA) production. In flask culture, specific GA productivities per dry weight biomass using dextrin and indigestible dextrin were 11.0 and 56.1 mU/mg-DW, respectively. Indigestible dextrin was a poor substrate for enzymatic hydrolysis. Rates of glucose formation from dextrin and indigestible dextrin by enzymatic hydrolysis were 0.477 and 0.100 mg-glucose/ml/h, respectively. For this reason, residual glucose concentrations in batch cultures grown on indigestible dextrin remained below 1.32 mg/ml where glucose-limiting conditions were easily maintained. Batch culture using indigestible dextrin had the same residual glucose profile as dextrin fed-batch culture, and nearly the same GA activity was obtained after 42.5 h of growth. However, between 42.5 and 66 h, the GA production rate of the indigestible dextrin batch culture (11.5 mU/ml/h) was higher than that of the dextrin fed-batch culture (6.5 mU/ml/h). During this period, a high amount of residual maltooligosaccharide was detected in the culture supernatant grown on indigestible dextrin. The high GA productivity observed in the indigestible dextrin batch culture may have resulted from the absence of glucose and the simultaneous presence of maltooligosaccharides throughout growth.     

Production of lipase by high cell density fed-batch culture of Candida cylindracea

Candida cylindracea NRRL Y-17506 was grown to produce extracellular lipase from oleic acid as a carbon source. Through flask cultures, it was found that the optimum initial oleic acid concentration for cell growth was 20 g l⁻¹. However, high initial concentrations of oleic acid up to 50 g l⁻¹ were not inhibitory. The highest extracellular lipase activity obtained in flask culture was 3.0 U ml⁻¹ after 48 h with 5 g l⁻¹ of initial oleic acid concentration. Fed-batch cultures (intermittent and stepwise feeding) were carried out to improve cell concentration and lipase activity. For the intermittent feeding fed-batch culture, the final cell concentration was 52 g l⁻¹ and the extracellular lipase activity was 6.3 U ml⁻¹ at 138.5 h. Stepwise feeding fed-batch cultures were carried out to simulate an exponential feeding and to investigate the effects of specific growth rate (0.02, 0.04 and 0.08 h⁻¹) on cell growth and lipase production. The highest final cell concentration obtained was 90 g l⁻¹ when the set point of specific growth rate (μ_set) was 0.02 h⁻¹. High specific growth rate (0.04 and 0.08 h⁻¹) decreased extracellular lipase production in the later part of fed-batch cultures due to build-up of the oleic acid oversupplied. The highest extracellular lipase activity was 23.7 U ml⁻¹ when μ_set was 0.02 h⁻¹, while the highest lipase productivity was 0.31 U ml⁻¹ h⁻¹ at μ_set of 0.08 h⁻¹.     

High-level production of arachidonic acid by fed-batch culture of <Emphasis Type="Italic"> Mortierella alpina</Emphasis> using NH<Subscript>4</Subscript>OH as a nitrogen source and pH control

Mortierella alpina was grown in a fed-batch culture using a 12-l jar fermenter with an initial 8-l working volume containing 20 g glucose l⁻¹ and 10 g corn-steep powder l⁻¹. Glucose was intermittently fed to give 32 g l⁻¹ at each time. The pH of culture was maintained using 14% (v/v) NH₄OH, which also acted as a nitrogen source. A final cell density of 72.5 g l⁻¹ was reached after 12.5 days with a content of arachidonic acid (ARA) at 18.8 g l⁻¹. These values were 4 and 1.8 times higher than the respective values in batch culture. Our results suggest that the combined feeding of glucose and NH₄⁺ to the growth of M. alpina could be applied for the industrial scale production of ARA.