Kinetics and modeling of GM-CSF production by recombinant yeast in a three-phase fluidized bed bioreactor

Continuous production of a recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) by Saccharomyces cerevisiae strain XV2181 (a/a, Trp 1) containing plasmid palphaADH2 and immobilized on porous glass beads in a fluidized bed bioreactor was studied. Kinetic models for plasmid stability, cell growth, and protein production in the three-phase fluidized bed bioreactor were developed and used to study the effects of solid loading or cell immobilization on plasmid stability and recombinant protein production. With increasing cell immobilization or solid loading in the bioreactor, plasmid stability and protein production improved significantly. The improvements could be attributed to the decreased theta value, which is the plasmid loss probability during cell division and is an indication of segregational instability of the recombinant cell, and the increased alpha value, which is the ratio of the specific growth rate of a plasmid-carrying cell to that of a plasmid-free cell and is indicative of competitive stability of the recombinant cell culture. theta decreased from 0.552 to 0.042 and alpha increased from 0.351 to 0.991 when solid loading in the bioreactor was increased from 5% (v/v) to 33%. The model simulation also showed that the specific growth rate of cells in the bioreactor was lower at higher solid loading. This indicated that there was significant mass transfer limitation, particularly for oxygen transfer, when the total cell density in the bioreactor was high at high solid loading. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 470-477, 1997.     

Kinetics of cell growth and cyclosporin A production by Tolypocladium inflatum when scaling up from shake flask to bioreactor

The kinetics of cell growth and Cyclosporin A (Cyc A) production by Tolypocladium inflatum were studied in shake flasks and bioreactors under controlled and uncontrolled pH conditions. In the case of the shake flask, the production time was extended to 226 h and the maximal antibiotic concentration was 76 mg/l. When scaling up the cultivation process to a bioreactor level, the production time was reduced to only 70 h with a significant increase in both the cell growth and the antibiotic production. The maximal dry cell weights in the case of the controlled pH and uncontrolled pH cultures in the bioreactor were 22.4 g/l and 14.2 g/l, respectively. The corresponding maximal dry cell weight values did not exceed 7.25 g/l with the shake flask cultures. The maximal values for Cyc A production were 144.72 and 131.4 mg/l for the controlled and uncontrolled pH cultures, respectively. It is also worth noting that a significant reduction was observed in both the dry cell mass and the antibiotic concentration after the Cyc A production phase, whereas the highest rate of antibiotic degradation was observed in the stirred tank bioreactor with an uncontrolled pH. Morphological characterization of the micromorphological cell growth (mycelial/pellet forms) was also performed during cultivation in the bioreactor.     

搅拌式生物反应器悬浮培养水母雪莲细胞的研究

应用 2L通气搅拌式生物反应器一步批式培养水母雪莲细胞。采用倾斜式搅拌桨代替透平桨 ,研究了搅拌转速、通气量和接种量对细胞生长和黄酮合成的影响 ,发现在 75r min、70 0～1000L min和 4.0～ 5.0gDCW L接种量下细胞生长和黄酮合成比较好。经过 12d培养细胞干重达 13.8gDCW L ,黄酮产量 416mg L ,黄酮含量占细胞干重的 30%。水母雪莲细胞生长及黄酮合成的进程表明 ,黄酮积累与细胞生长呈正相关。对细胞聚集体分布的研究发现 ,流变压力使细胞聚集体分裂 ,使反应器中细胞生长受到影响 ,黄酮产量较摇瓶中降低     

Production of Manganese Peroxidase by free pellets ofPhanerochaete chrysosporium in an Expanded-Bed Bioreactor

Summary Continuous production of Manganese Peroxidase by free pellets ofPhanerochaete chrysosporium in an Expanded-Bed Bioreactor was successfully achieved for more than 30 days of operation. The stability in continuous production was attained because of two modifications in the operational policy: an adequate feed control and the supply of oxygen in a pulsing mode. Feed control allowed to stablish an equilibrium between primary and secondary metabolism, thus avoiding metabolic stress. The use of pulsation had two beneficial effects: 1) Increase of stability by avoiding bed compactation during operation performance; 2) Control of mycelial pellet size. The bioreactor operated without oxygen pulsation did not maintain enzyme production longer than 14 days.     

Production of a secreted glycoprotein from an inducible promoter system in a perfusion bioreactor

The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated.     

Continuous production of manganese peroxidase by Phanerochaete chrysosporium immobilized on polyurethane foam in a pulsed packed-bed bioreactor

The bottleneck of the application of manganese peroxidase (MnP) on an industrial scale in pulp biobleaching or in degradation of hazardous compounds is the lack of an efficient production system. Three main problems arise for the continuous production of MnP during secondary metabolism of Phanerochaete chrysosporium: enzyme production occurs only under specific physiological conditions corresponding to C or N limitation, high O(2) tension, and adequate Mn(+2) concentration; the enzyme that is produced is destabilized by extracellular proteases; and excessive growth of the mycelium blocks effective oxygen transfer. To overcome these drawbacks, continuous production of MnP was optimized by selecting a suitable bioreactor configuration and the environmental and operating conditions affecting both enzyme production and stability. The combination between a proper feed rate and the application of a pulsation in a packed-bed bioreactor permitted the maintenance of continuous secretion of MnP while limiting mycelial growth and avoiding bed clogging. Environmental factors as an Mn(+2) concentration of 5000 muM and high oxygen tension enhanced MnP production. The hydraulics of the bioreactor corresponding to a plug flow model with partial mixing and an operating hydraulic rentention time of 24 h were optimal to achieve stable operating conditions. This policy allowed long operation periods, obtaining higher productivities than the best reported in the literature. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 130-137, 1997.     

Continuous antibiotic production by an immobilized cyanobacterium in a seaweed-type bioreactor

The filamentous cyanobacterium,Scytonema sp. TISTR 8208, which produces a cyclic peptide antibiotic, was cultivated for 20 d in a seaweed-type bioreactor containing anchored polyurethan foam strips. Cells immobilized onto the foam strips produced the antibiotic for only several days, and the secreted antibiotic disappeared very rapidly from the medium. Cells accumulated the antibiotic intracellularly in a growth-related manner, and secreted it in the stationary phase. Since the antibiotic has a stable physico-chemical nature, the cells seem to take it up and metabolize it. When continuous cultivation was attempted, stable production of the antibiotic was maintained in the bioreactor for 16 d at a dilution rate of 0.01 h^–1. Three times more antibiotic was produced in the continuous culture than in the batch culture by the 16th day.     

Inoculum production of the ectomycorrhizal fungus Pisolithus microcarpus in an airlift bioreactor

Many important tree species in reforestation programs are dependent on ectomycorrhizal symbiosis in order to survive and grow, mainly in poor soils. The exploitation of this symbiosis to increase plant productivity demands the establishment of inoculum production methods. This study aims to propose an inoculum production method of the ectomycorrhizal fungus Pisolithus microcarpus (isolate UFSC-Pt116) using liquid fermentation in an airlift bioreactor with external circulation. The fungus grew as dark dense pellets during a batch fermentation at 25.5 degrees C and air inlet of 0.26-0.43 vvm. The maximum biomass (dry weight) achieved in the airlift bioreactor was approximately 5 g.l(-1) after 10-11 days. The specific growth rate (micro(x)) in the exponential phase was 0.576 day(-1), the yield factor (Y(X/S)) 0.418, and the productivity (P(X)) 0.480 g.l(-1).day(-1). This specific growth rate was higher than that observed by other authors during fermentation processes with other Pisolithus isolates. The method seems to be very suitable for biomass production of this fungus. However, new studies on the fungus growth morphology in this system, as well as on the efficiency of the process for the cultivation of other ectomycorrhizal fungi, are necessary. It is also necessary to test the infectivity and efficiency of the inoculum towards the hosts.     

Investigation of acid proteinase biosynthesis by the fungus Humicola lutea 120-5 in an airlift bioreactor

Acid proteinase production using filamentous fungus Humicola lutea 120-5 was studied under batch and continuous fermentation conditions in an airlift bioreactor. A comparison with proteinase production by fungal cells, cultivated in stirred tank bioreactor was made. The process performance in both fermentation devices was similar with respect to substrate utilization, biomass, and enzyme concentration. Continuous acid proteinase production was achieved for 14 days at an optimal dilution rate of 0.05/h with maximum specific activity of 90 U/mg DW of mycelia and yield of 38 U/mg glucose. The volumetric productivity (50 U/ml. h) was approximately 3 times higher than this of the batch system. All continuous experiments were carried out without any bacterial contamination, due to the low pH (3.0-3.5) during the process. The "pellet" type growth of the fungus in the airlift reactor prevented the system from plugging with filaments.     

Effect of support materials on antibiotic MSW2000 production by immobilized Streptomyces violatus

The production of an antibiotic by free and immobilized cells of Streptomyces violatus through entrapment or adsorption on different materials was investigated. S. violatus entrapped in Ca-alginate beads gave low antibiotic activity compared to the free cell or adsorbed cell, while the adsorption of S. violatus on sponge cubes yielded the highest antibiotic concentration after 4 days of incubation in static cultures. A starch concentration of 10 g/L was optimum for the production of the antibiotic by adsorbed cells. The weight and size of the sponge cubes used for immobilization influenced production of the antibiotic and the optimum weight and size of the sponge were 0.8 g and 1.0 cm(3), respectively, yielding a maximum antibiotic production of 280 mg/ml. Maximum antibiotic production was obtained at an initial pH value of 7.5 and in an inoculum size of 3 ml (spore suspension) per 50 ml. The production of the antibiotic in a fixed-bed bioreactor reached a maximum value after 2 days of incubation at a circulation rate of 30 ml/h. The immobilized cells in the bioreactor were reused seven successive times over a period of 14 days.     

Application of factorial design to the optimization of medium composition in batch cultures of Streptomyces lividans TK21 producing a hybrid antibiotic

Summary The composition of the liquid medium employed to obtain a hybrid antibiotic in batch cultures of a recombinant strain of Streptomyces lividans TK21 has been studied. Starch and glutamic acid are the most appropriate carbon and nitrogen sources to support respectively cell growth and antibiotic production. A central composite experimental design has been employed to derive a statistical model of the effect of phosphate and glutamic acid on growth and antibiotic production, and an initial concentration of 10 mM phosphate and 52.8 mM glutamic acid have been found optimal to maximize the final antibiotic concentration in batch cultures.     

Batch and continuous ribonuclease production by immobilized Aspergillus clavatus cells in a bubble-collumn bioreactor

Summary Ribonuclease production using immobilized cells (IC) of Aspergillus clavatus has been studied under batch, repeated-batch and continuous fermentation conditions in a bubble-column bioreactor and compared with production by free cells, Immobilization was achieved by the method of cryostructurization in polyvinyl alcohol beads. The effect of various aeration rates in a column bioreactor has been investigated. Enzyme production by IC [42 000 units (U)·l^–1] during batch fermentations was comparable to that of a free-cell system. The specific productivity of IC was 8.5 times higher than that of free cells. In repeated batch fermentation at various aeration rates, successful reuse of IC was obtained, with comparable levels of enzyme production. Continuous ribonuclease production was achieved for 44 days at 1 vvm aeration and a dilution rate of 0.0 h^–1 with volumetric productivity (450 U·1^–1) and yield.     

Periodic operation of immobilized live cell bioreactor for the production of candicidin

A lumped model for cell growth and secondary metabolite production in an immobilized live cell bioreactor has been developed. This model is applied here to simulate the performance of an immobilized bioreactor under steady-state conditions and under conditions of periodically varying concentration of a growth-limiting substrate. The results of the simulation study were experimentally verified in the case of the production of the antibiotic candicidin by Streptomyces griseus in an immobilized bioreactor with forced periodic operation. The results of the studies suggest that periodically operated immobilized live cell bioreactors can provide a potent alternative for the production of non-growth-associated biochemicals, as compared to free cell fermentations, pulsed fermentations with process cycle regeneration, and nonregenerated bioreactors. This work has demonstrated that by frequent pulsing of the growth limiting nutrient, stable extended production can be obtained at high specific cellular productivities.     

Dynamic change of metabolites and nutrients in suspension cells of <Emphasis Type="Italic">Panax Quinquefolium</Emphasis> L. in bioreactor

In order to understand how the nutrient elements were taken up during the cell growth as well as the production of metabolites, it was quite necessary to identify the dynamic change of metabolites and nutrients in suspension cells of Panax quinquefolium in bioreactor. In this study, dynamic accumulation of biomass and ginsenosides Re, Rb1 and polysaccharide as well as major nutrients consumption in cell suspension culture of P. quinquefolium in a 5-L stirred tank bioreactor were investigated. The dry cell weight and the contents of ginsenosides Re, Rb1 and polysaccharide reached the maximum peak simultaneously on about 21 days and the results showed that cell growth and metabolites synthesis related to nutrients consumption. For this reason, we supposed that the contents of metabolites can be increased through added nutrient at the right moment. These results provided theory reference for two-stage or continuous perfusion culture in suspension cells of P. quinquefolium in bioreactor.     

林肯链霉菌合成林可霉素代谢调节的研究

在摇瓶条件下研究了葡萄糖、铵盐、磷酸盐对林可霉素产生菌林肯链霉菌的生长及林可霉素生物合成的影响。发酵过程中林可霉素的合成主要发生在菌体生长期,逐渐下降。使用6%的葡萄糖未发现通常所说的“葡萄糖效应”。0.2%铵盐有利于细胞生长,但0.8%NH+4对林可霉素的生物合成具有抑制作用。发酵48h后补加0.6% NH^＋_４,对林可霉素的生成没有显著影响。0.05%～0.1%磷酸盐对林可霉素合成具有较强的抑制作用。并就磷酸盐对菌体由初级代谢转向次级代谢的作用作了初步考察。     

Production of actinomycin D with immobilizedStreptomyces parvullus under nitrogen and carbon starvation conditions

Summary The productivity of actinomycin D withStreptomyces parvullus could be enhanced with calcium alginate immobilized cells and a synthetic starvation medium in a continuous air-lift bioreactor with moderately low dilution rate. In addition, the cell mass must be cycled periodically between the growth and starvation media to maintain long-term antibiotic production.     

Production of Choristoneura fumiferana Nucleopolyhedrovirus in C. fumiferana (CF-2C1 Cells) in a 3 Litre Bioreactor Using Serum-free Medium

The purpose of this study was to develop a cell culture process in a bioreactor for the production of a viral insecticide for the spruce budworm, Choristoneura fumiferana . Several cell lines were tested for their growth in serum-free medium suspension cultures. One cell line, CF-124T-2C1 (CF-2C1), was successfully adapted to grow in suspension cultures in SFM. Serum-free Ex-Cell 405 medium produced a much higher cell density (6.3 x 10 6 cells ml -1 ) than the Grace's medium supplemented with 10% fetal bovine serum (2.5 x 10 6 cells ml -1 ). Also, a higher yield of virus was obtained in the former medium. Ex-Cell 405, was used to study the growth of CF-2C1 cells and the production of C. fumiferana nucleopolyhedrovirus (CfMNPV) in a 3 l bioreactor. Under these conditions, a specific growth rate ( μ) of 0.027 h -1 was obtained during the exponential growth phase, and the specific carbon dioxide evolution rate, as determined by on-line measurement, was 0.9 x 10 -16 mol cell -1 s -1 and 1.78 x 10 -16 mol cell -1 s -1 during growth and infection phases, respectively. Virus production in bioreactor cultures infected at 1.3 x 10 6 cells ml -1 was consistently lower than that obtained in Erlenmeyer shake flasks. Only 26% of the cells were infected in the bioreactor compared to 44% in the shake flasks. However, a higher yield of occluded virus was obtained in the bioreactor cultures than in shake flasks. The production of occlusion bodies (OB) achieved in bioreactor cultures was 2 x 10 6 OB ml -1 .     

Feasibility of culturing C2C12 mouse myoblasts on glass microcarriers in a continuous stirred tank bioreactor

Commercial culturing of mammalian cell lines is increasing in importance as more biological products unique to mammals are being produced in genetically altered mammalian cells. Most mammalian cells are anchorage dependent, so they must be cultured on a support matrix. This limitation, along with the requirement of a low shear environment, severely effects the scale-up of bench-scale culture systems. The need to culture mammalian cells on a support matrix limits the increase in cell population to a factor of 10-20 before growth virtually stops due to contact inhibition. Commercial culturing systems for anchorage dependent cells are batch processes because of the combination of contact inhibition and support matrix requirements. Development of a continuous bioreactor system could allow both unlimited scale-up and continuous cell-mass production. To design a continuous reactor, a mathematical model to predict the reactor performance should be developed. This paper addresses the development of a mathematical model for predicting continuous bioreactor performance. It was found that anchorage dependent C2C12 mouse myoblast cells, a continuous cell line, followed Monod kinetics for glucose consumption and cell mass production in batch flask experiments, with w_max = 0.040 hr^у and K_m = 2.5 mM. Furthermore, it was found that these parameters could be used to predict the glucose consumption in a continuous bioreactor operated with constant feed of seeded microcarriers operated at two different residence times. The success of this model implies the possibility of developing a continuous cell harvesting and reinoculation system using a microcarrier bioreactor to produce cell mass.     

Ajmalicine production by cell cultures of Catharanthus roseus: from shake flask to bioreactor

The productivity of a cell culture for the production of a secondary metabolite is defined by three factors: specific growth rate, specific product formation rate, and biomass concentration during production. The effect of scaling-up from shake flask to bioreactor on growth and production and the effect of increasing the biomass concentration were investigated for the production of ajmalicine by Catharanthus roseus cell suspensions. Growth of biomass was not affected by the type of culture vessel. Growth, carbohydrate storage, glucose and oxygen consumption, and the carbon dioxide production could be predicted rather well by a structured model with the internal phosphate and the external glucose concentration as the controlling factors. The production of ajmalicine on production medium in a shake flask was not reproduced in a bioreactor. The production could be restored by creating a gas regime in the bioreactor comparable to that in a shake flask. Increasing the biomass concentration both in a shake flask and in a stirred fermenter decreased the ajmalicine production rate. This effect could be removed partly by controlling the oxygen concentration in the more dense culture at 85% air saturation.     

Continuous cultivation of a human intestinal microflora at high cell concentration via controlled culture recycle

A bioreactor system equipped with a hollow fiber cross-filtration module was used for continuous cultivation of Lactobacillus acidophilus at high cell concentrations. The growth rate did not correlate with the lactate concentration if the residual glucose concentration was kept nearly zero in the culture broth. To achieve this, an effective control method of medium feed rate was developed on the basis of the correlation between the specific glucose consumption rate (nu) and the specific cell growth rate (mu), i.e. nu = 52.90 mu + 0.39. Growth up to 50 g dry wt l-1 was achieved without glucose accumulation under the total cell recycle. Via the partial cell recycle, continuous biomass production was achieved with a steady-state L. acidophilus concentration and dilution rate being 40 gl-1 and 0.09 h-1. © Rapid Science Ltd. 1998