Repeated Batch Culture Research Articles

Pectin lyase production by Penicillium griseoroseum grown in sugar cane juice in repeated batch cultures

Penicillium griseoroseum cultured in the presence of sucrose and yeast extract produces pectin lyase (EC 4.2.2.10) (PL) in the absence of its natural inducer pectin. This fungus was cultured in a fermenter at an aeration rate of 0.5 l/min for the first 25 h and 1.0 l/min for the remainder of the culture period, and at a stirring rate of 200 rev/min for the entire culture period. Fungal spores were inoculated directly into the fermenter at a final concentration of 5 × 104 spores/ml. The fungus was cultured in minimal medium supplemented with powdered dehydrated sugar cane juice, producing PL without added yeast extract. Maximum PL activity (0.067 IU/ml) was obtained after 65 h in batch culture. Pellet morphology of the mycelia made it possible to carry out three cycles of repeated batch culture. The same medium was used for renewal as for the single batch culture. The initial cycle was 53 h, after which approximately 0.103 IU/ml of PL was obtained. After this period, the medium was renewed and fermentation continued for two more cycles, which lasted approximately 20 h. Activity of PL obtained in the second cycle was approximately 0.118 IU/ml and in the third, approximately 0.109 IU/ml.

DEGRADATION OF DIMETHYLPHTHALATE BY CELLS OF BACILLUS SP. IMMOBILIZED IN CALCIUM ALGINATE AND POLYURETHANE FOAM

A Bacillus sp. which is capable of degrading dimethylphthalate (DMP) was immobilized in calcium alginate and polyurethane foam for efficient and long term degradation of DMP. Freely suspended cells (1012 cfu ml−1) degraded a maximum of 20 mM DMP. Whereas, alginate-(1012 cfu g−1 beads) and polyurethane foam-entrapped (0.34 × 106–9 cfu g−1 foam cubes) cells degraded a maximum of 40 mM DMP within 12–15 days of incubation. Polyurethane foam-entrapped cells degraded 30 mM of DMP at 4 days and alginate-entrapped cells degraded within 10 to 12 days of incubation irrespective of the cell population. When the initial concentration of DMP increased to 50 mM, the DMP degrading ability of the immobilized cells was not increased even after 20 days. Repeated batch cultures by alginate-entrapped cells with initial 35 mM DMP loading could be reused for a maximum of 20 cycles. However, the degradation rate was gradually decreased when the beads were reused for more than 15 cycles. On the other hand, the foam-entrapped cells, with the same initial DMP loading there was no decrease in DMP degrading ability and could be reused for more than 20 cycles. The packed bed reactor with alginate-entrapped cells (1 × 1010–12 cfu g−1 bead) could be continuously operated for 7–8 days with an initial 25 mM DMP at a flow rate of 50 ml h−1. Whereas, the polyurethane foam-entrapped cells (1 × 106–9 cfu g−1 foam cubes) could be operated continuously for more than 90 days with the same initial DMP loading at a flow rate of 100 ml h−1. Thus the enhanced degradation of DMP could be achieved by immobilizing the cells of Bacillus sp. in calcium alginate and polyurethane foam as compared to that of freely suspended cells.

Characterization of the stability of recombinant protein production in the GS-NS0 expression system.

The GS-NS0 system is an important mammalian expression system used largely within industry for the high-level expression of recombinant proteins for therapeutic use. It is essential that the productivity of this system remains stable throughout culture expansion for the successful long-term production of recombinant proteins. Here we present a study of the stability of recombinant protein production from unamplified GS-NS0 cell lines over extended period of continuous culture. The cell lines used in this study were generated by the transfection of NS0 cells with DNA encoding for a secreted recombinant protein and by two subsequent rounds of limiting dilution cloning prior to analysis of stability. The stability of recombinant protein production was assessed at intervals over a period of 134 days using repeated batch culture in shake flasks. Heterogeneous stability was identified. The productivity of some clones remained consistent throughout 134 days of continuous culture. Others exhibit rapid and progressive loss of productivity. Analysis of the causal relationships underlying stability indicates that the initial transfectant determines the susceptibility to loss or retention of productivity. Selection of production clones on the basis of growth and productivity alone will not predict stability during long-term culture. Our research indicates that stable high-producing clones can readily be obtained from use of the GS-NS0 system in the absence of amplification but there may be molecular features of the original transfectants that could serve as very important predictive indicators of the stability of recombinant protein production.

Enhanced anthocyanin production by repeated-batch culture of strawberry cells with medium shift.

Repeated-batch cultures of strawberry cells (Fragaria ananassa cv. Shikinari) subjected to four medium-shift procedures (constant LS medium, constant B5 medium, alternation between LS and B5 starting from LS and alternation between LS and B5 starting from B5) were investigated for the enhanced anthocyanin productivity. To determine the optimum period for repeated batch cultures, two medium-shift periods of 9 and 14 days were studied, which represent the end of the exponential growth phase and the stationary phase. By comparison with the corresponding batch cultures, higher anthocyanin productivity was achieved for all the repeated-batch cultures at a 9-day medium-shift period. The average anthocyanin productivity was enhanced 1.7- and 1.76-fold by repeated-batch cultures in constant LS and constant B5 medium at a 9-day shift period for 45 days, respectively. No further improvement was observed when the medium was alternated between LS (the growth medium) and B5 (the production medium). Anthocyanin production was unstable at a 14-day shift period regardless of the medium-shift procedures. The results show that it is feasible to improve anthocyanin production by a repeated-batch culture of strawberry cells.

Efficient production of cellulolytic and xylanolytic enzymes by the rumen anaerobic fungus, Neocallimastix frontalis, in a repeated batch culture

In a batch culture of Neocallimastix frontalis in a medium (pH 6.8) containing 8 g· l −1 cellulose, the concentrations of the fermentation products and the cellulolytic and xylanolytic enzymes did not increase in comparison with those of cultures in a medium containing 4 g· l −1 cellulose. Therefore, kinetic studies were performed to determine the effect that products such as acetate, formate, lactate and ethanol have in inhibiting the growth. The reduction of the specific growth rate by the fermentation products could be expressed by using a noncompetitive inhibition model, and it was found that at low concentrations, acetate was the strongest inhibitor among the inhibitory products studied. In order to reduce the inhibition by the fermentation products, a repeated batch culture was carried out whereby none of the fatty acids exceeded 50 mM. In this repeated batch culture, xylanase, endoglucanase, β-glucosidase, and avicelase were continuously produced in the medium during cultivation, and 18200, 4550, 3790 and 129 IU g· l −1 of these enzymes, respectively, were produced up to 20 d of culture.

Efficient Production of Cellulolytic and Xylanolytic Enzymes by the Rumen Anaerobic Fungus, Neocallimastix frontalis, in a Repeated Batch Culture.

In a batch culture of Neocallimastix frontalis in a medium (pH 6.8) containing 8 g.l(-1) cellulose, the concentrations of the fermentation products and the cellulolytic and xylanolytic enzymes did not increase in comparison with those of cultures in a medium containing 4 g.l(-1) cellulose. Therefore, kinetic studies were performed to determine the effect that products such as acetate, formate, lactate and ethanol have in inhibiting the growth. The reduction of the specific growth rate by the fermentation products could be expressed by using a noncompetitive inhibition model, and it was found that at low concentrations, acetate was the strongest inhibitor among the inhibitory products studied. In order to reduce the inhibition by the fermentation products, a repeated batch culture was carried out whereby none of the fatty acids exceeded 50 mM. In this repeated batch culture, xylanase, endoglucanase, beta-glucosidase, and avicelase were continuously produced in the medium during cultivation, and 18200, 4550, 3790 and 129 IU g.l(-1) of these enzymes, respectively, were produced up to 20 d of culture.

Microbial hydrogen production from sweet potato starch residue

Clostridium butyricum could produce hydrogen from a sweet potato starch residue upon supplementation of nitrogen sources. A repeated batch culture using a mixed culture of C. butyricum and Enterobacter aerogenes produced hydrogen with a yield of 2.4 mol H 2/mol glucose under a controlled culture pH of 5.25 in a medium consisting of the sweet potato starch residue and 0.1% Polypepton without addition of any reducing agents. Rhodobacter sp. M-19 produced hydrogen from the supernatant of the culture broth obtained in the repeated batch culture containing C. butyricum and E. aerogenes when 50 μg/ l Na 2MoO 4·2H 2O and 20 mg/ l EDTA were added to the supernatant and it was cultured under a controlled culture pH of 7.5. A high yield of hydrogen of 7.0 mol H 2/mol glucose from the starch remaining in the starch residue was attained in two-step repeated batch cultures containing C. butyricum and E. aerogenes, and by Rhodobacter sp. M-19.

Microbial Hydrogen Production from Sweet Potato Starch Residue.

Clostridium butyricum could produce hydrogen from a sweet potato starch residue upon supplementation of nitrogen sources. A repeated batch culture using a mixed culture of C. butyricum and Enterobacter aerogenes produced hydrogen with a yield of 2.4 mol H2/mol glucose under a controlled culture pH of 5.25 in a medium consisting of the sweet potato starch residue and 0.1% Polypepton without addition of any reducing agents. Rhodobacter sp. M-19 produced hydrogen from the supernatant of the culture broth obtained in the repeated batch culture containing C. butyricum and E. aerogenes when 50 microg/l Na2MoO4.2H2O and 20 mg/l EDTA were added to the supernatant and it was cultured under a controlled culture pH of 7.5. A high yield of hydrogen of 7.0 mol H2/mol glucose from the starch remaining in the starch residue was attained in two-step repeated batch cultures containing C. butyricum and E. aerogenes, and by Rhodobacter sp. M-19.

Enhanced and prolonged production of plantlets regenerated from carrot callus in a viscous additive-supplemented medium

To reduce the hydrodynamic stress in plant cell culture and enhance the production of regenerated plantlets, a liquid medium containing a viscous additive was newly designed and plantlet production from embryogenic carrot callus cultivated in the medium was examined. Na-alginate or carboxymethyl cellulose (CMC) was used as the viscous additive. The viscosity of the medium increased with increasing additive content and the number of regenerated plantlets also increased. When carrot calli were cultivated in the medium containing 0.4% CMC, designated as N medium (viscosity, 3 mPa·-s), the maximum enhancement of plantlet regeneration, approximately 2.5 times higher than that in the control medium, was obtained. Enlargement of callus size observed in N medium is considered to be the main reason for the enhanced plantlet regeneration. Regeneration enhancement was sufficiently induced after calli were cultivated once in N medium, but this regenerarion ability rapidly disappeared after once cultivation in the conventional medium. In repeated batch culture using N medium, plantlet production continued at a high level for 18 batches (250 d) with no significant decrease, while in the control culture without CMC the number of plantlets produced dropped to almost zero by the sixth batch (84 d).

Enhanced and Prolonged Production of Plantlets Regenerated from Carrot Callus in a Viscous Additive-Supplemented Medium.

To reduce the hydrodynamic stress in plant cell culture and enhance the production of regenerated plantlets, a liquid medium containing a viscous additive was newly designed and plantlet production from embryogenic carrot callus cultivated in the medium was examined. Na-alginate or carboxymethyl cellulose (CMC) was used as the viscous additive. The viscosity of the medium increased with increasing additive content and the number of regenerated plantlets also increased. When carrot calli were cultivated in the medium containing 0.4% CMC, designated as N medium (viscosity, 3 mPa.s), the maximum enhancement of plantlet regeneration, approximately 2.5 times higher than that in the control medium, was obtained. Enlargement of callus size observed in N medium is considered to be the main reason for the enhanced plantlet regeneration. Regeneration enhancement was sufficiently induced after calli were cultivated once in N medium, but this regeneration ability rapidly disappeared after once cultivation in the conventional medium. In repeated batch culture using N medium, plantlet production continued at a high level for 18 batches (250 d) with no significant decrease, while in the control culture without CMC the number of plantlets produced dropped to almost zero by the sixth batch (84 d).

Immobilization of fungus Aspergillus sp. by a novel cryogel technique for production of extracellular hydrolytic enzymes

Aerobic cells of a fungus isolate Aspergillus sp. CX-1 have been immobilized in macroporous cryoPAG and in different composite cryoPAGs — fibrous adjunct carriers. The productivity of the extracellular enzymes (exo-1.4-β-glucanase, endo-1.4-β-glucanase, β-glucosidase and xylanase), and the viability, growth and ultrastructure of the immobilized fungus have been studied. The enzyme activities and stability during long-term repeated batch cultivation in the immobilized fungus were higher than in free mycelia when batch cultivated. The fungus immobilized in the composite cryoPAG, containing polypropylene non-woven fabric, possessed the highest exo-1.4-β-glucanase activity, the longest durability of enzyme production (85 days) and the most reliable mechanical strength. The fungus immobilized in porous composite cryogel possessed a variety of advantages including easy control of cryogel porosity, improved mechanical strength and durability, simplicity of construction, high enzyme productivity and high stability.

Mathematical modeling of citric acid production by repeated batch culture.

A mathematical model has been created for the process of citric acid biosynthesis by yeast (mutant strain Yarrowia lipolytica) cultivated by the repeated batch (RB) method on ethanol under conditions of nitrogen limitation. The model accounts for cell growth as a function of nitrogen concentration in the culture liquid; nitrogen uptake by growing cells; citric acid production; pH control in the fermentor by means of NaOH addition; and changes in system volume. The model represents a system of five nonlinear differential equations. Experimental measurements of cell concentration, citric acid concentration, and cultivation broth volume were used with the least squares method to determine the values of eight model parameters. The parameter values obtained were consistent with literature data and general concepts of cell growth and citric acid biosynthesis. The model has been used to predict optimum RB culture conditions.

Decreased chimeric antibody productivity of KR12H-1 transfectoma during long-term culture results from decreased antibody gene copy number

The stability of KR12H-1 transfectoma in regard to chimeric antibody production was examined during long-term, repeated batch culture without selection pressure using antibiotics. Both serum-supplemented and serum-free media were used. Regardless of the medium used, the specific antibody productivity (q(Ab)) of transfectoma decreased by 60% to 88% during 70-day culture. This loss of antibody productivity was not due mainly to the appearance of a nonproducing population (NP) of transfectoma. The percentage of a producing population (P), which was monitored by the limiting dilution method, remained over 90% until the end of culture, indicating that the q(Ab) of P decreased during the culture. Flow cytometric data also showed the increase of cell population with low fluorescence intensity during culture, indicating that the intracellular antibody content of P decreased. The subclones of P obtained at the end of long-term culture were further characterized. Compared with the q(Ab) of P at the beginning of long-term culture, the q(Ab) of most P subclones was significantly low, confirming that the loss of antibody productivity was due mainly to the decreased q(Ab) of P during long-term culture. The decreased antibody gene copy number of P subclones was found to be partly responsible for the decreased q(Ab) of P during long-term culture. (c) 1996 John Wiley & Sons, Inc.

Ethanol production by repeated batch culture using yeast cells immobilized within porous cellulose carriers

Ethanol was efficiently produced for 20 d by repeated batch culture using yeast cells immobilized in porous cellulose carriers. The physical structure of the carrier beads, which had small pores distributed on the outer surface and relatively large pores distributed in the interior, was found to be effective for cell immobilization. The effects of chemical modifications to the basic cellulose carrier on cell immobilization and ethanol production were also examined in repeated batch cultures. The chemical modifications had little effect on either cell immobilization or ethanol production.

Aggregate formation of rCHO cells and its maintenance in repeated batch culture in the absence of cell adhesion materials

Aggregate formation of recombinant Chinese hamster ovary (rCHO) cells capable of producing granulocyte colony-stimulating factor (G-CSF), using medium lacking cell adhesion materials in a repeated batch culture, was examined together with cell growth, cell viability and G-CSF production. The rCHO culture was conducted in a rotary shaker and the medium was changed every five days. The formation of stable cell aggregates with high reproducibility was observed after the first medium change. The size of the cell aggregates (consisting of several 10s to 40,000 cells) formed during the repeated batch culture ranged from 30 to 600 μm. The cell density of the aggregates reached as high as 2 × 10 6 cells/ml and the viability was maintained at more than 80% for 19 d. Changing the medium to avoid glucose exhaustion effectively maintained the cell density, cell viability and G-CSF productivity at high levels.

Immobilization of alkaliphilic Bacillus sp. cells for xylanase production using batch and continuous culture.

Agar-immobilized alkaliphilic Bacillus sp. AR-009 cells were used for xylanase production using batch and continuous culture. In a batch culture, maximum enzyme production was observed after 48 h and remained high up to 72 h. In repeated batch cultivation, immobilized cells produced an appreciable level of xylanase activity in seven consecutive batches without any significant decline in productivity. For continuous xylanase production, immobilized cells were packed in a jacketed glass column and sterile medium was continuously pumped. A stable continuous production of xylanase was observed over a period of 1 mo. The volumetric productivity of the continuous culture was 17-fold higher than the batch culture using free cells.

Ethanol Production by Repeated Batch Culture Using Yeast Cells Immobilized within Porous Cellulose Carriers.

Ethanol Production by Repeated Batch Culture Using Yeast Cells Immobilized within Porous Cellulose Carriers.

Synergistic effect of active oxygen species and alginate on chitinase production by Wasabia japonica cells and its application

The specific chitinase productivity of a Wasabia japonica cell suspension culture under pure oxygen aeration was 3.8 times higher than that of a suspension culture aerated with ordinary air. During aeration with pure oxygen, both oxygen consumption by the cells and the H 2O 2 concentration in the medium increased. Addition of H 2O 2 to the cultivation medium also promoted the specific chitinase productivity. H 2O 2 could pass freely through the cell membrane. It was assumed that the excess oxygen was converted into active oxygen species such as H 2O 2, and that the promotion of chitinase production was probably due to the generated active oxygen species. Addition of alginate oligomer (AO, an endogenous elicitor-like substance) to cultures aerated with pure oxygen or supplemented with H 2O 2 resulted in synergistic increases in chitinase production. Based on these results, the development of a simple and efficient chitinase production system was investigated. Cells were immobilized in alginate gel (instead of adding AO to the medium) and cultivated in a medium containing H 2O 2. The specific chitinase productivity increased to the levels observed in the suspension culture system. During repeated batch cultivation of immobilized cells, the chitinase production remained stable for three repeated batches. When immobilized protoplasts were cultivated in a medium containing H 2O 2, there was 7-fold increase in chitinase production compared with that of immobilized cells.

Development of chitosan-magnetite aggregates containing Nitrosomonas europaea cells for nitrification enhancement

A cell suspension of the nitrifying bacterium Nitrosomonas europaea obtained after 96-h cultivation was subjected to magnetic separation using chitosan-conjugated magnetite particles (chitosan-magnetite), which have the ability to form aggregates with microbial cells. An equilibrium condition was obtained at room temperature after 30 min and over 90% of the cells were recovered when the chitosan-magnetite concentration was 200 mg/ l. The relationship between the cell concentration in the supernatant in equilibrium and the number of cells adsorbed per 1g chitosan-magnetite was expressed by a Freundlich-type adsorption equation. A high nitrifying bacterium activity yield was obtained with a chitosan-magnetite concentration between 100 and 200 mg/ l. Repeated batch culture resulted in more N. europaea cells accumulating on the aggregates and as a consequence their nitrification activity improved further. The chitosan-magnetite/cell aggregates were recovered and employed to remove ammonia from artificial wastewater together with PVA-alginate gel beads containing the denitrifying bacterium Paracoccus denitrificans. A higher ammonia removal rate was achieved under aerobic conditions in comparison with that obtained when N. europaea and P. denitrificans were coimmobilized in PVA-alginate gel beads.

Estimation of cell damage in bench- and pilot-scale affinity expanded-bed chromatography for the purification of monoclonal antibodies

The first step in downstream processing of mammalian cell culture includes the separation of the cells without cell damage to avoid the release of intracellular enzymes, which could potentially cause proteolytic degradation of the target protein and may increase the impurities for further chromatographic steps. This especially includes the reduction of host DNA for therapeutic proteins. The aim of this investigation was to examine the extent of cell damage at the bench and pilot scale using a stabilized fluidized bed (expanded bed) for direct recovery of IgG from cell culture broth. For this purpose, Streamline-25 and -200 columns containing 75 mL and 5 L of rProtein A matrix, respectively, were used. The repeated batch cultivations resulted in high cell viabilities of about 90% prior purification. The pH was gently adjusted to pH 8 before the broth was applied to the gel. In bench scale, 1 to 6 L of unclarified feed was applied to the Streamline-25 column. In pilot scale, up to 95 L was processed using the Streamline-200 column. The antibodies from 95 L of unclarified feed were recovered after approximately 1.5 h. The possible cell damage, caused either by the equipment or by the cells' passage through the expanded bed, was detected by the following assays: microscopic count of the cells using trypan blue dye exclusion to determine viability; monitoring of intracellular components (i.e., DNA concentration); activity of lactate dehydrogenase (LDH); and, finally, the particle load in the flow through and the eluate. Despite the sensitivity of hybridoma cells to shear forces, neither the high flow rate (300 to 450 cm/h) nor the passage of the cells through the expanded bed caused any relevant cell damage or clogging of the gel. Excellent DNA depletion was observed.