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 .     

Production and biotransformation of 6-pentyl-α-pyrone by Trichoderma harzianum in two-phase culture systems

The final concentration of 6-pentyl-a-pyrone (6PP) produced in cultures of Trichoderma spp. is limited by the fact that inhibition of biomass growth occurs at 6PP concentrations as low as 100 mg/l. The aim of this work was to evaluate liquid-liquid extractive fermentation systems as an alternative to overcome the toxicity problems and to increase the production of 6PP by this fungus. Two alkanes (n-decane and n-hexadecane) and two dicarboxylic esters (dibutyl phthalate and dioctyl phthalate) were evaluated in shake flask cultures. The highest 6PP production (173 ppm) was achieved when n-hexadecane was used, being 3.5-fold the maximum 6PP concentration of a culture without the solvent. Cultivation of Trichoderma harzianum in a 10-1 bioreactor with n-hexadecane yielded 6PP production ninefold higher than that from control cultures. However, 6PP production in the bioreactor (83 ppm) was lower than in shake flasks. Differences in the power drawn to the fluid at each scale could account for such behavior. Even in the presence of the solvent, 6PP content decreased after reaching its maximal concentration.     

Novel bioreactors for the culture and expansion of aggregative neural stem cells

Neural stem cells have been cultured as three-dimensional aggregates in a number of different types of bioreactors. The design and configuration of the bioreactor are shown to be crucial factors for the successful propagation of the cells. A novel bioreactor with liquid re-circulation and a working volume of 200 ml has been designed, tested and shown to be able to produce a higher cell vitality compared to those produced in multi-well plates, shake flasks and stirred flasks. The novel reactor was able to produce a total density of cells of 3.5 x 10(6) cells/ml consisting of a larger number of smaller and proliferative aggregates, compared to only 1.8 x 10(6) cells/ml produced in a multi-well plate. Shake flasks and stirred flasks commonly used for facilitating mass transfer in the culture of micro-organisms are shown to be unsuitable for the propagation of neural stem cells.     

溶氧对Blakeslea trispora分批发酵生产β-胡萝卜素的影响

在摇瓶和5 L发酵罐中研究了溶氧 (DO) 对Blakeslea trispora分批发酵生产β-胡萝卜素的影响,总结了5 L发酵罐中β-胡萝卜素发酵过程中溶氧的变化规律.结果表明,当500 mL摇瓶装液量为50 mL,转速为240 r/min条件下发酵生产β-胡萝卜素产量最大,达到3.416 g/L; 5 L发酵罐中,在搅拌转速为1 000 r/min,通气量为1.5 vvm的条件下,β-胡萝卜素的产量可达到3.712 g/L,略高于摇瓶,这可能是由于5 L发酵罐中的气液传递和混合状况好于摇瓶,促进了产物的合成.     

Scale-up of biopesticide production processes using wastewater sludge as a raw material

Studies were conducted on the production of Bacillus thuringiensis (Bt)-based biopesticides to ascertain the performance of the process in shake flasks, and in two geometrically similar fermentors (15 and 150 l) utilizing wastewater sludge as a raw material. The results showed that it was possible to achieve better oxygen transfer in the larger capacity fermentor. Viable cell counts increased by 38–55% in the bioreactor compared to shake flasks. As for spore counts, an increase of 25% was observed when changing from shake flask to fermentor experiments. Spore counts were unchanged in bench (15 l) and pilot scale (5.3–5.5 e⁺⁰⁸ cfu/ml; 150 l). An improvement of 30% in the entomotoxicity potential was obtained at pilot scale. Protease activity increased by two to four times at bench and pilot scale, respectively, compared to the maximum activity obtained in shake flasks. The maximum protease activity (4.1 IU/ml) was obtained in pilot scale due to better oxygen transfer. The Bt fermentation process using sludge as raw material was successfully scaled up and resulted in high productivity for toxin protein yield and a high protease activity.     

Production of carbonyl reductase by Metschnikowia koreensis

A new strain of the yeast Metschnikowia koreensis was grown in shake flasks and a stirred bioreactor for the production of carbonyl reductase. The optimal conditions in the bioreactor for maximizing the biomass specific activity of the enzyme were found to be: a medium composed of glucose (20 g/L), peptone (5 g/L), yeast extract (5 g/L) and zinc sulfate (0.3g/L); the pH controlled at 7; the temperature controlled at 25 °C; an agitation speed of 500 rpm; and an aeration rate of 0.25 vvm. In the bioreactor, a biomass specific enzyme activity of 115.6 U/gDCW was obtained and the maximum biomass concentration was 15.3 gDCW/L. The biomass specific enzyme activity obtained in the optimized bioreactor culture was 11-fold higher than the best result achieved in shake flasks. The bioreactor culture afforded a 2.7-fold higher biomass concentration than could be attained in shake flasks.     

Modeling of growth and laccase production by Pycnoporus sanguineus

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L^?1 days^?1, or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L^?1.     

The physical characterisation of a microscale parallel bioreactor platform with an industrial CHO cell line expressing an IgG4

There is a growing body of evidence that the ambr™ workstation from TAP Biosystems performs well in terms of helping to select appropriate clones for scale-up studies. Here we have investigated the physical characteristics of this microscale bioreactor system and found that these are quite different from those that exist in larger scale stirred bioreactors. For example, the flow regime in the ambr™ vessel is transitional rather than turbulent and the sparged air/oxygen superficial gas velocity is relatively very low whilst the specific power input is much higher (~400 W/m³) when compared to that used at larger scales (typically ~20 W/m³). This specific power input is necessary in order to achieve k_La values sufficiently high to satisfy the oxygen demand of the cells and control of dO₂. In line with other studies, we find that the culture of CHO cells in a 15 mL ambr™ bioreactor gave similar cell growth and productivity to that achieved in a 5 L stirred bioreactor whilst the results from shake flasks were significantly different. Given the differences in physical characteristics between the ambr™ and larger stirred bioreactors, we suggest that this similarity in biological performance is due to their similar control capabilities and the ‘equivalence of the stress parameters’ across the scales when compared with shake flasks.     

Improvement of rifemycins production by Amycolatopsis mediterranei in batch and fed-batch cultures

The production of rifamycins B and SV using glucose as main C-source by Amycolatopsis mediterranei in batch and fed-batch culture was investigated. Fed-batch culture using glucose as mono feeding substrate either in the form of pulse addition, in case of shake flask, or with constant feeding rate, in bioreactor level, proved to be an alternative production system with a significant increase in both volumetric and specific antibiotic production. The maximal concentrations of about 1146 mg/l and 2500 mg/l of rifamycins B and SV, respectively, was obtained in fed-batch culture in bioreactor level under non-oxygen limitation. On the other hand, the rate of rifamycins production was increased from 6.58 to 12.13 mg/l x h for rifamycin B and from 9.47 to 31.83 mg/l x h for rifamycin SV on the bioprocess transfer and improvement from the conventional batch cultivation in shake flask to fed-batch cultivation in stirred tank bioreactor.     

Cultivation of transgenicNicotiana tabacum suspension cells in bioreactors for the production of mGM-CSF

TransgenicNicotiana tabacum cells were cultivated for the production of murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in both a stirred, tank biore|actor and an airlift bioreactor with draft tube. Cell growth and mGM-CSF production in the airlift bioreactor were found to be better than those achieved in the stirred tank bioreactor. In the airlift bioreactor. 9.0 g/L of cells and 2.2 ng/mL of mGM-CSF were obtained (11.0 g/L and 2.4 ng/mL, respectively in shake flasks). Although the lag period was prolonged and mGM-CSF production was lowered by 33% in the stirred tank bioreactor as compared to the control culture, the maximum cell density was increased up to 12.0 g/L due to better mixing by agitation at the higher cell density.     

Comparison of Cell Growth and Alkaloid Production of Catharanthus roseus Cells Cultured in Shake Flask and in Bioreactor

The cell growth and alkaloid production of Catharanthus roseus (L.) G. Don cells cultured in the shake flasks with different volumes and in the stirred tank bioreactor (10 L) were compared. Cell dry weight and alkaloid production showed no significant difference in the small volume scale-up shake flasks. When more broths were added to a certain volume in the shake flask, both cell weight and alkaloid production were decreased. The maximum cell dry weight was similar between the cell cultures in the shake flask and the bioreactor, but the alkaloid production of cells was much less in the bioreactor. Gas regime and shear stress were recognized to be the main factors contributing the important effect on alkaloid production during the scale-up processes.     

A study of oxygen transfer in shake flasks using a non-invasive oxygen sensor

We describe a study of oxygen transfer in shake flasks using a non-invasive optical sensor. This study investigates the effect of different plugs, presence of baffles, and the type of media on the dissolved oxygen profiles during Escherichia coli fermentation. We measured the volumetric mass transfer coefficient (k(L)a) under various conditions and also the resistances of the various plugs. Finally, we compared shake flask k(L)a with that from a stirred tank fermentor. By matching k(L)a's we were able to obtain similar growth and recombinant protein product formation kinetics in both a fermentor and a shake flask. These results provide a quantitative comparison of fermentations in a shake flask vs. a bench-scale fermentor and should be valuable in guiding scale-up efforts.     

Azadirachtin production by hairy root cultivation of Azadirachta indica in a modified stirred tank reactor

Present investigation involves hairy root cultivation of Azadirachta indica in a modified stirred tank reactor under optimized culture conditions for maximum volumetric productivity of azadirachtin. The selected hairy root line (Az-35) was induced via Agrobacterium rhizogenes LBA 920-mediated transformation of A. indica leaf explants (Coimbatore variety, India). Liquid culture of the hairy roots was developed in a modified Murashige and Skoog medium (MM2). To further enhance the productivity of azadirachtin, selected growth regulators (1.0?mg/l IAA and 0.025?mg/l GA₃), permeabilizing agent (0.5?% v/v DNBP), a biotic elicitor (1?% v/v Curvularia (culture filtrate)) and an indirectly linked biosynthetic precursor (50?mg/l cholesterol) were added in the growth medium on 15th day of the hairy root cultivation period in shake flask. Highest azadirachtin production (113?mg/l) was obtained on 25th day of the growth cycle with a biomass of 21?g/l DW. Further, batch cultivation of hairy roots was carried out in a novel liquid-phase bioreactor configuration (modified stirred tank reactor with polyurethane foam as root support) to investigate the possible scale-up of the established A. indica hairy root culture. A biomass production of 15.2?g/l with azadirachtin accumulation in the hairy roots of 6.4?mg/g (97.28?mg/l) could be achieved after 25?days of the batch cultivation period, which was ~27 and ~14?% less biomass and azadirachtin concentration obtained respectively, in shake flasks. An overall volumetric productivity of 3.89?mg/(l?day) of azadirachtin was obtained in the bioreactor.     

Effect of shear stress on anthraquinones production by Rubia tinctorum suspension cultures

Suspension cultures of Rubia tinctorum, an anthraquinones (AQs) producer, were grown both in Erlenmeyer flasks at 100 rpm and in a 1.5 L mechanically stirred tank bioreactor operating at 450 rpm. The effect of hydrodynamic stress on cell viability, biomass, and AQs production was evaluated. Cell viability showed a transient decrease in the bioreactor during the first days, returning to the initial values toward the end of the culture time. The biomass obtained in the bioreactor was 29% lower than that attained in the Erlenmeyer flasks. The H2O2 production in the bioreactor (with peaks at 7 and 10 days) was about 15 times higher than that obtained in the flasks. A clear relationship exists between the maximum concentration of H2O2 generated and AQs produced. The AQs content in the bioreactor was 233% higher than that in the Erlenmeyer flasks. The AQs specific productivity in the stirred tank and in the Erlenmeyer flasks was 70.7 and 28.5 micromol/g FW/day, respectively. This production capability was maintained in the regrowth assays. On the other hand, the negative effects of hydrodynamic stress on viability and biomass concentration observed in the bioreactor culture were reverted in the regrowth cultures. It can be concluded that R. tinctorum suspension cultures are able to grow in stirred tanks at 450 rpm responding to the hydrodynamic stress with higher concentrations of AQs, which suggest the possibility of a technological approach taking advantage of this phenomenon.     

Exopolysaccharide production by Acremonium persicinum in stirred-tank and air-lift fermentors

Summary Exopolysaccharide production by the fungus Acremonium persicinum was affected by the culture system used. The yields achieved in shake flasks were not obtained in a stirred tank reactor, except at very low stirring speeds (100 rpm). However when grown in an air-lift fermentor, exopolysaccharide levels were similar to those found with shake flask cultures. Results suggest that both dissolved oxygen tension and shear rate may determine the ability of this organism to synthesise this exopolysaccharide. Offprint requests to: R. J. Seviour     

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.     

Effect of bioreactor configuration on the growth and maturation of Picea sitchensis somatic embryo cultures

Somatic embryo suspension cultures of Picea sitchensis (Sitka spruce) derived from two cell lines, SS03 and SS10, were grown in shake flasks, air-lift, bubble, stirred tank and hanging stirrer bar bioreactors. Cell line SS03 yielded freely suspended and individual stage 1 embryos, while the embryos of SS10 were present in large aggregates. Compared to shake flasks, proliferation in bioreactors resulted in increased biomass; however, cell line morphology influenced the effect of different bioreactor configurations on growth and maturation of embryo cultures. Somatic embryos grown in shake flasks and bioreactors were matured on gelled solid medium and in submerged culture where gelled solid medium was covered with a layer of liquid medium. The number of stage 3 (mature) embryos produced from SS03 in the bubble bioreactor was significantly higher than those from stirred tank and hanging stirrer bar bioreactors with both solid medium and submerged culture. Submerged culture was unsuitable for SS10 embryo maturation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of aggregate size in cell cultures of<Emphasis Type="Italic"> Saussurea medusa</Emphasis> on cell growth and jaceosidin production

Cell suspension cultures of Saussurea medusa were grown in shake flasks and a 5-l stirred tank bioreactor. Biomass and jaceosidin distribution in cell aggregates of different sizes were investigated during the cultivation period. The results showed that on day 10, jaceosidin accumulation showed an increase with increasing size of the cell aggregate to 4 mm in diameter, with the highest jaceosidin accumulation being 12.2 mg/g. An inverse tendency was observed with cell aggregates larger than 4 mm in diameter, with the lowest accumulation being 3.1 mg/g. However, all of the cell aggregates, despite their size, synthesized almost the same amount of jaceosidin at day 12. Oxygen diffusion limitation and cell-cell contact may explain this behavior. In comparison with cells cultivated in shake flasks, decreased biomass and decreased jaceosidin concentration were observed when the cells were cultivated in a stirred tank bioreactor. The sublytic effects caused by the hydrodynamic stress in combination with insufficient nutrients in the bioreactor may cause cell damage.     

Enhanced catharanthine production in catharanthus roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors

Chemical and fungal elicitors were added to Catharanthus roseus cell suspension cultures so as to improve the production of indole alkaloids. A synergistic effect on alkaloid accumulation was observed in C. roseus cell cultures when treated with some combined elicitors of fungal preparations and chemicals. Among them, the combination of tetramethyl amminium bromide and Aspergillum niger mycelial homogenate gave the highest ajmalicine yield (63 mg l(-1)) and an improved catharanthine accumulation (17 mg l(-1)). The combined elicitors of malate and sodium alginate resulted in the highest catharanthine yield (26 mg l(-1)) and a high ajmalicine accumulation (41 mg l(-1)) in the cell cultures. Based on the synergistic effect of malate and sodium alginate, a process with enhanced catharanthine production in Catharanthus roseus cell cultures was developed in shake flasks and a bioreactor. After 10 days of culture, 25 mg l(-1), 32 mg l(-1) and 22 mg l(-1) catharanthine yield were obtained in 500-ml flasks, 1000-ml flasks and in a 20-l airlift bioreactor, respectively. Upon malate-alginate combining treatments, peroxidase, catalase and superoxide dismutase activities decreased in elicited cells but phenylalanine ammonia lyase and lipoxygenase activities increased dramatically. That suggests a typical defense responses took place in the combined elicitors-treated cell cultures. Furthermore, the combined elicitors also caused a significant increase of malondialdehyde level in cell cultures, which suggests a serious lipid peroxidation occurred in the elicited cell cultures. Comparison of these results suggests that malate and alginate combining treatment also stimulates defense responses, such as lipid peroxidation, in all C. roseus culture processes and this may mediate the indole alkaloid production via jasmonate pathway.     

Statistical medium optimization and production of a hyperthermostable lipase from Burkholderia cepacia in a bioreactor

AIM: Statistical medium optimization for maximum production of a hyperthermostable lipase from Burkholderia cepacia and its validation in a bioreactor. METHODS AND RESULTS: Burkholderia cepacia was grown in shake flasks containing 1% glucose, 0.1% KH2PO4, 0.5% NH4Cl, 0.24% (NH4)2HPO4, 0.01% MgSO4.7H2O and 1% emulsified palm oil, at 45 degrees C and pH 7.0, agitated at 250 rev min(-1) with 6-h-old inoculum (2% v/v) for 20 h. A fourfold enhancement in lipase production (50 U ml(-1)) and an approximately three fold increase in specific activity (160 U mg(-1)) by B. cepacia was obtained in a 14 litre bioreactor within 15 h after statistical optimization following shake flask culture. The statistical model was obtained using face centred central composite design (FCCCD) with five variables: glucose, palm oil, incubation time, inoculum density and agitation. The model suggested no interactive effect of the five factors, although incubation period, inoculum and carbon concentration were the important variables. CONCLUSIONS: The maximum lipase production was 50 U ml(-1), with specific activity 160 U mg(-1) protein, in a 14 litre bioreactor after 15 h in a medium obtained after statistical optimization in shake flasks. Further, the model predicted reduction in time for lipase production with reduction in total carbon supply. SIGNIFICANCE AND IMPACT OF THE STUDY: Statistical optimization allows quick optimization of a large number of variables. It also provides a deep insight into the regulatory role of various parameters involved in enzyme production.