Scale up production of isoflavonoids in cell suspension cultures of Pueraria tuberosa grown in shake flasks and bioreactor

Cell cultures of Pueraria tuberosa were grown in vessels of different sizes and 2L stirred tank bioreactor containing modified MS medium with morphactin (0.1 mg l^?1) and 2iP (5.0 mg l^?1) and 20% inoculum. Stable growth and total isoflavonoid yield of 76.6 mg l^?1 were recorded in the cultures during scale up. This was in concordance with the persistent yield of the individual isoflavonoids regardless of the vessel size.     

Improved guggulsterone production from sugars, precursors, and morphactin in cell cultures of Commiphora wightii grown in shake flasks and a bioreactor

Cell cultures of Commiphora wightii (Arnott.) Bhandari were grown in shake flasks and a bioreactor and an increase in guggulsterone accumulation up to 18 μg l⁻¹ was recorded in cells grown in the production medium containing a combination of sucrose:glucose (4% total), precursors (phenylalanine, pyruvic acid, xylose, and sodium acetate), morphactin, and 2iP. A yield of 10 g l⁻¹ biomass and ∼200 μg l⁻¹ guggulsterone was recorded in a 3-l flask and in a 2-l stirred tank bioreactor compared with 6.6 g biomass and 67 μg l⁻¹ guggulsterone in 250-ml flasks. Increased vessel size was correlated with increased biomass and guggulsterone accumulation. 2iP alone was not effective for biomass and guggulsterone accumulation in cell cultures of C. wightii.     

Isoflavone production in Cyclopia subternata Vogel (honeybush) suspension cultures grown in shake flasks and stirred-tank bioreactor

Suspension cultures of the endemic South-African plant Cyclopia subternata were established for the first time and evaluated for the presence of isoflavones. The influence of light, as well as medium supplementation strategies with phenylalanine, casein hydrolysate and coconut water on biomass growth and isoflavone production were examined. The highest levels of 7-O-β-glucosides of calycosin, pseudobaptigenin and formononetin (275.57, 125.37 and 147.28 mg/100 g DW, respectively) were recorded for cultures grown in the absence of light, whereas coconut water substantially promoted biomass growth. Cell suspensions were subsequently grown in the 2-l stirred-tank bioreactor. Maximum productivity of 7-O-β-glucosides of calycosin, pseudobaptigenin and formononetin (0.96, 0.44 and 0.22 mg l^?1 day^?1, respectively) in bioreactor-cultivated cells was obtained for biomass grown in the dark and supplemented with coconut water. The results indicate that C. subternata suspension cultures can be utilised for the production of the specified isoflavone derivatives absent in the intact plant.     

Ethrel treatment enhanced isoflavonoids accumulation in cell suspension cultures of Pueraria tuberosa, a woody legume

The cell cultures of Pueraria tuberosa, a perennial leguminous lianas, were maintained in modified MS medium (KNO₃ 475 mg l⁻¹, thiamine 1 mg l⁻¹, biotin 1 mg l⁻¹, calcium pantothenate 1 mg l⁻¹) containing 0.1 mg l⁻¹ 2,4,5-trichloroacetic acid and 0.1 mg l⁻¹ kinetin. Isoflavonoids (puerarin, genistin, daidzein, genistein) accumulation in cell suspension cultures was increased by 14-fold to ~12 mg l⁻¹ after 48 h of adding 100 μM ethrel. Ethrel inhibitors (silver nitrate and silver thiosulfate) completely inhibited this effect in the presence of ethrel and isoflavonoids were not detected in the spent medium. The increase was dose dependent and can be explored to trigger high yield of isoflavonoids production.     

In situ pH maintenance for mammalian cell cultures in shake flasks and tissue culture flasks

pH in animal cell cultures decreases due to production of metabolites like lactate. pH control via measurement and base addition is not easily possible in small‐scale culture formats like tissue‐culture flasks and shake flasks. A hydrogel‐based system is reported for in situ pH maintenance without pH measurement in such formats, and is demonstrated to maintain pH between 6.8 and 7.2 for a suspension CHO cell line in CD CHO medium and between 7.3 and 7.5 for adherent A549 cells in DMEM:F12 containing 10% FBS. This system for pH maintenance, along with our previous report of hydrogels for controlled nutrient delivery in shake flasks can allow shake flasks to better mimic bioreactor‐based fed batch operation for initial screening during cell line and process development for recombinant protein production in mammalian cells. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Methyl jasmonate effect on diterpenoid accumulation in Salvia sclarea hairy root culture in shake flasks and sprinkle bioreactor

Hairy root cultures of Salvia sclarea were grown in shake flasks and 10 L nutrient sprinkle bioreactor, running for 30 days and the effects of methyl jasmonate (MJ) on their growth and capacity to accumulate diterpenoids were measured. We found that MJ concentration and exposure time to the elicitor were factors that strongly affected the diterpenoid production. The highest diterpenoid accumulation (67.5 ± 7.1 mg g⁻¹ dry weight, calculated as a sum of ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone) without reduction of biomass, was achieved when the 23-day-old hairy roots in bioreactor culture were exposed to 125 μM MJ for 7 days. The roots produced 9 and 3.8 times as much aethiopinone (40 ± 5.9 mg g⁻¹ dry weight) and salvipisone (12.6 ± 0.4 mg g⁻¹ dry weight), respectively, as roots cultured in shake flasks. Our results imply that cultivation of S. sclarea hairy roots in sprinkle bioreactor after elicitation with MJ may be valuable to enhance production of the bioactive diterpenoids.     

The production and antiprotozoal activity of abietane diterpenes in Salvia austriaca hairy roots grown in shake flasks and bioreactor

The biomass and concentration of bioactive quinone methide-type diterpenes in hairy roots of Salvia austriaca were determined and compared with levels of these metabolites in roots of field-grown plants. The cultures were maintained in shake flasks and a nutrient sprinkle bioreactor. Diterpene production was more efficient in the shake flask root culture than the bioreactor one. Biomass and diterpene production within the shake flask culture was evaluated using Schenk and Hildebrandt (SH), Gamborg (B5), and woody plant medium (WPM), with both full- and half-strength macro and micronutrient concentrations (1/2 SH, 1/2 B5, and 1/2 WPM). Among the tested media, SH medium proved to be most effective for biomass and diterpene production. In this medium, the transformed roots accumulated the levels of taxodone (3.89?mg?g^?1 DW; equivalent to 63.3?mg?L^?1), taxodione (1.15?mg?g^?1 DW; equivalent to 17.4?mg?L^?1), 15-deoxy-fuerstione (2.15?mg?g^?1 DW; equivalent to 32.5?mg?L^?1), and 7-(2′-oxohexyl)-taxodione (0.076?mg?g^?1 DW; equivalent to 1.1?mg?L^?1). Three diterpenes were also detected in the roots of S. austriaca intact plants, but their concentrations were lower than those in hairy root culture. No 7-(2′-oxohexyl)-taxodione was found in the roots of field-grown plants. The hairy roots were able to maintain high metabolite levels even for 6 years of cultivation. Taxodone, taxodione, 15-deoxy-fuerstione, and 7-(2′-oxohexyl)-taxodione were tested for in vitro activity against Trypanosoma brucei rhodesiense, T. cruzi, and Plasmodium falciparum and their cytotoxicity was determined using L6 cells. Among these compounds, taxodione was the most active against T. brucei rhodesiense [IC₅₀?=?0.05?µM with high selectivity, selectivity index (SI)?=?38]. Taxodione was found to inhibit the growth of P. falciparum and T. cruzi by 50% at respective concentrations of 1.9 and 7.1?µM (SI values of 1.0 and 0.27). Other diterpenoids demonstrated weaker activity against tested parasites (IC₅₀ values ranging from 0.62 to 194.7?µM) and lower selectivity (SI value ranged from 0.4 to 5.0).     

Effect of sugar concentration on ginsenoside biosynthesis in hairy root cultures of Panax quinquefolium cultivated in shake flasks and nutrient sprinkle bioreactor

The study assessed the influence of sugar concentration (10, 20, 30, 50, 70, 100, 120 g l^?1) on growth and ginsenoside biosynthesis in Panax quinquefolium hairy roots cultivated in shake flasks and a nutrient sprinkle bioreactor. The highest growth rate was achieved in medium containing 3–5 % sucrose. More than 70 g l^?1 or less than 20 g l^?1 sugar content in the medium induces significant inhibition of root growth when cultivated in shake flasks. The saponin content was determined using HPLC. The maximum yield (above 9 mg g^?1 d.w.) of the sum of six examined ginsenosides (Rb₁, Rb₂, Rc, Rd, Re and Rg₁) in hairy roots cultivated in shake flasks was obtained with 30 g l^?1 sucrose in the medium. The sucrose concentration in the medium was found to correlate with saponin content in bioreactor-cultured specimens. A higher level of protopanaxadiol derivatives was found for lower (20 and 30 g l^?1) sucrose concentrations; higher sucrose concentrations (50 and 70 g l^?1) in the medium stimulated a higher level of Rg group saponins.     

Culture medium optimization for improved puerarin production by cell suspension cultures of Pueraria tuberosa (Roxb. ex Willd.) DC.

The effects of carbon, nitrogen, phosphate, and copper on cell growth and production of the isoflavone puerarin by suspension cultures of Pueraria tuberosa (Roxb. ex. Willd.) DC were investigated. Among the various sugars evaluated (glucose, galactose, fructose, maltose, and sucrose), use of sucrose in the medium led to the maximum accumulation of puerarin. A sucrose-feeding strategy in which additional sucrose was added to the flasks 15?d into the culture cycle stimulated both cell biomass and puerarin production. The maximum production of puerarin was obtained when a concentration balance of 20:60?mM NH ₄ ⁺ /NO ₃ ^? was used as the nitrogen source. Alteration in the concentration balance of nitrogen components (NH ₄ ⁺ /NO ₃ ^? 60:20?mM) or the use of either NH ₄ ⁺ or NO ₃ ^? alone decreased biomass production and puerarin accumulation compared with the control culture (NH ₄ ⁺ /NO ₃ ^? 20:20?mM). High amounts of phosphate (2.5 and 5?mM) in the medium inhibited puerarin production whereas 0.625?mM phosphate promoted puerarin production (68.3???g/g DW on day?25). An increase in Cu²⁺ concentration from 0.025 to 0.05?mg/l in the P. tuberosa cell culture medium resulted in a 2.2-fold increase in puerarin production (up to 141???g/g DW on day?25) but reduced cell culture biomass.     

Interaction of cultural conditions and end-product distribution in Bacillus subtilis grown in shake flasks

Summary A culture of Bacillus subtilis, in which the relative production of acetoin (Ac) and butanediol (Bu) is highly sensitive to oxygen tension as well as to mixing conditions, was used to evaluate several culture conditions in 500-ml shake flasks. The concentration ratio of these metabolites (Ac/Bu) produced in a defined period of culture time was used as a parameter for comparative purposes. The influence of working volume, shaking speed, broth viscosity and the presence of baffles were evaluated. Using unbaffled flasks it was found that working volume had the most influence on oxygenation in shake flasks, especially below 10%, where differences in Ac/Bu ratios up to ten times could be measured. Shaking speed played an important role only at values higher than 400 rpm or when small working volumes were used. The addition of xanthan gum decreased the Ac/Bu ratio nearly four times under equivalent working conditions and also diminished the influence of shaking speed. In general, Ac/Bu was higher when sulphite oxygen transfer rate (OTR) values were higher. However, the test culture was able to detect differences which were not evident using the OTR method. Comparing Ac/Bu ratios in stirred fermentors from the literature, it seems that similar oxygenation conditions can be reached in non-baffled shake flasks only at very high shaking speeds using small working volumes. With baffled flasks, our data suggest that better oxygenation and mixing can be achieved in shake flasks if compared with those obtained in stirred fermentors at conventional power inputs.     

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.     

Morphological development and gibberellin production by different strains of Gibberella fujikuroi in shake flasks and bioreactor

Strain H-984 of G. fujikuroi grown for 38h in a shake flask with medium containing 20g glucose l^–1, 3g yeast extract l^–1, 2.5g NH4NO3 l^–1, 0.5g KH2PO4 l^–1, 0.1g MgSO4 l^–1, 1g CaCO₃ l^–1, and inoculated into a bioreactor with medium containing 60g glucose l^–1; 1g NH_4Cl l^–1; 3g KH2PO4 l^–1 and 1.5g MgSO₄ l^–1 produced 1100mg gibberellic acid l–1.     

Studies on production of ajmalicine in shake flasks by multiple shoot cultures of Catharanthus roseus

The effects of different concentrations of indole-3-acetic acid (IAA) and benzyladenine (BA) on production of ajmalicine by multiple shoot cultures of Catharanthus roseus (C. roseus) were studied. By supplementing Murashige and Skoog's (MS) medium with a high concentration of IAA (11.42 microM) and a low concentration of BA (2.22 microM), shoot cultures accumulated high levels of ajmalicine. When culture medium was fortified with a low concentration of IAA (2.85 microM) and a high concentration of BA (8.90 microM), shoots released high levels of ajmalicine into the culture medium. Quantification of ajmalicine was performed by high performance liquid chromatography (HPLC). The highest concentration of ajmalicine production (0.166% dry wt) was obtained by shoot cultures grown in MS medium containing IAA (11.42 microM) on 20 days of cultivation. Shoot cultures accumulated ajmalicine 4.2-fold more in IAA (11.42 microM) supplemented medium compared with the high concentration of BA (8.90 microM). The content of ajmalicine concentration in the medium was quantified. Shoot cultures grown in BA (8.90 microM) supplemented medium released the maximum production of ajmalicine (0.853 g/L) into the culture medium after 15 days of cultivation. The experimental data show that the secretion of ajmalicine was 2-fold more into the culture medium supplemented with a high concentration of BA compared to that with a low concentration of BA. Data presented here show that production of ajmalicine by shoot cultures is not correlated with growth rate. Dimeric indole alkaloids vincristine and vinblastine were not present in shoot cultures. Ajmalicine production by shoot cultures was 2.4-fold higher compared to leaves of 1-year-old naturally grown plants.     

Synergistic effect of morphactin on cytokinin-induced production of isoflavonoids in cell cultures of Pueraria tuberosa (Roxb. ex. Willd.) DC

Isoflavonoids, the functional molecules of Fabaceae, are under clinical trials against cancer, osteoporosis and cardiovascular diseases. In this study, the efficacy of different plant growth regulators was evaluated for optimizing the production of isoflavonoids in Pueraria tuberosa. The cultures were maintained in Murashige and Skoog’s medium containing 0.1 mg l⁻¹ 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 0.1 mg l⁻¹ kinetin. The addition of 5.0 mg l⁻¹ N⁶-(2-Isopentenyl) adenine (2iP) resulted in about ∼32-folds increase in production of isoflavonoids, while about ∼23-folds increase was recorded in the absence of kinetin in the maintenance medium. A maximum yield of isoflavonoids (∼80 mg l⁻¹; 82-folds increase) was obtained in cultures grown at 0.1 mg l⁻¹ morphactin and 5.0 mg l⁻¹ of 2iP. However, 2,4,5-T in combination with 2iP was ineffective for their production. Among different plant growth regulators tested, maximum yields of puerarin, genistin, daidzein and genistein were 17.4, 15.9, 69.0 and 0.04 mg l⁻¹, respectively. The study suggested that the presence of two cytokinins or 2iP with morphactin in the culture medium markedly enhanced the production of isoflavonoids in P. tuberosa.     

High stilbenes accumulation in root cultures of <Emphasis Type="Italic">Cayratia trifolia</Emphasis> (L.) Domin grown in shake flasks

The Root cultures of Cayratia trifolia (Vitaceae) a tropical lianas, were maintained in liquid Murashige and Skoog’s medium containing 0.5 mg l⁻¹ NAA, 0.1 mg l⁻¹ kinetin with 3% sucrose. These root cultures when grown with 6% sucrose accumulated stilbenes (piceid, resveratrol, viniferin, ampelopsin) in high amounts, which on elicitation by 500 mg l⁻¹ yeast extract, 50 μM salicylic acid (SA), 50 μM methyl jasmonate (MeJa), 500 μM ethrel added at 25th day, increased up to ninefolds (7.1 mg l⁻¹). Addition of alar or phenylalanine along with the elicitors further enhanced the stilbenes content. In the present study, stilbenes accumulation up to 12 folds (9.2 mg l⁻¹) was obtained with SA and alar. The SA was the most effective in increasing the stilbenes contents while less than control values were recorded in the cells treated with MeJa. The roots could be grown up to 2 l flasks. The present work demonstrates that presence of precursor and sucrose during elicitation at an appropriate time combined with growth retardation significantly increased the production of stilbenes in C. trifolia cell cultures.     

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.     

Inulinolytic activity of broths of Aspergillus niger ATCC 204447 cultivated in shake flasks and stirred tank bioreactor

It is the first detailed study of an inulinolytic fungus Aspergillus niger ATCC 204447 since its discovery, covering submerged cultivations both in shake flasks and a stirred tank bioreactor. Various carbon sources were applied to induce the inulinolytic activity in shake flask cultures. The highest volumetric and specific (per gram of biomass) activities (respectively 0.68 U/mL and 184 U g/X) were observed for the initial inulin and sucrose concentrations equal to 20 g/L. The fungus grew as large (>3 mm) spherical pellets. The influence of inoculum density and application of microparticle‐enhanced cultivation (MPEC) were studied in the batch bioreactor cultivations. Inoculum density moderately affected the inulinolytic activities, whose highest values were 0.7 U/mL and 165 U g/X at the lowest studied spore density of 3.33·10⁸ L^?1. Dispersed hyphae evolved in the bioreactor made the broth difficult to aerate due to high apparent viscosity (exceeding 200 Pa sⁿ at shear rate about 0.05 s^?1) and shear thinned properties (flow behavior index below 0.2). In MPEC (10 μm talc microparticles) the pellets of diameter between 1 and 2 mm were formed, which facilitated the aeration of the broth and increased the specific inulinolytic activity 3.5‐fold.     

Involvement of abscisic acid in ozone-induced puerarin production of Pueraria thomsnii Benth. suspension cell cultures

Exposure to ozone induced a rapid increase in the levels of the sesquiterpene phytohormone abscisic acid (ABA) and the isoflavone puerarin in suspension cell cultures of Pueraria thomsnii Benth. The observed increases in ABA and puerarin were dependent on the concentration of ozone applied to P. thomsnii cell cultures. In order to examine the role of ABA in ozone-induced puerarin production, cell suspensions were pretreated with the ABA biosynthetic inhibitor fluridone. Following ozone exposure, fluridone treatment suppressed ABA accumulation suggesting ABA was normally synthesized de novo through the carotenoid pathway. Fluridone also blocked ozone-induced puerarin production, which could be reversed through application of exogenous ABA. However, in the absence of ozone, ABA itself had no effect on puerarin accumulation in the suspension cells. Taken together, the data indicate that ozone is an efficient elicitor of puerarin production and may be particularly applicable for improving puerarin production in plant cell cultures. Furthermore, we demonstrate that ABA is one factor associated with ozone-induced puerarin production in P. thomsnii cell cultures.     

Broth rheology, growth and metabolite production of Beta vulgaris suspension culture: a comparative study between cultures grown in shake flasks and in a stirred tank

Cells of Beta vulgaris have the ability to grow in a stirred tank under an impeller tip speed as high as 95.3 cm seg⁻¹. Comparing this system with cultures performing in shake flasks, a decrease of the cell concentration, betalains production, and growth rate was observed. However, the kinetic profiles of aggregates size and cellular viability were practically the same. The cultures carried out in the fermentor showed a major accumulation of extracellular arabinogalactoprotein and polysaccharide, which is an indication of the cell response to hydrodynamic stress. These extracellular molecules produced a considerable change in the rheology of cell-free medium. This change in the rheology can be playing an important role in the reduction of the actual hydrodynamic stress during cultivation.     

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: from shake flasks to a 20-L bioreactor

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.