In sito galanthamine extraction during the cultivation of Leucojum aestivum L. shoot culture in two‐phase bubble column cultivation system

Two‐phase bioreactor cultivation system was developed and applied for in sito recovery of extracellular galanthamine during the cultivation of Leucojum aestivum L. shoot culture in a modified column bioreactor system. The inclusion of an external circulation column with adsorbent resin Amberlite XAD‐4 as a second phase, on the 21st day of the beginning of cultivation resulted in 1.25 folds increase in biomass accumulation and maximal amounts of accumulated galanthamine of 6 mg/L (3.1 mg/L intracellular and 2.9 mg/L extracellular). It was demonstrated that the inclusion of a second phase at the cultivation of the L. aestivum shoot culture in a bubble column bioreactor with internal sections redirected the alkaloid metabolism to galanthamine synthesis and inhibits the synthesis of hemanthamine and lycorine type alkaloids. Our research demonstrated that the application of the two‐phase cultivation systems could be an important tool to increase the yields of valuable secondary metabolites in plant tissue culture‐based bioprocess.     

Stimulating effect of both 4’‐O‐methylnorbelladine feeding and temporary immersion conditions on galanthamine and lycorine production by Leucojum aestivum L. bulblets

Bulb cultures of Leucojum aestivum and L. aestivum ‘Gravety Giant’ were subcultured in medium containing the precursor 4’‐O‐methylnorbelladine (MN) at various concentrations [0 (control), 0.15 and 0.3 g/L]. The cultures were conducted in bioreactor RITA® and lasted for 15, 30, 40 and 50 days. The growth rate and the alkaloid accumulation in bulblets were studied. For this latter purpose, a purification method was developed. It comprised a highly selective solid phase extraction using on the one hand, UPTI‐CLEAN SI and SCX cartridges for plant extracts and on the other hand, 2H cartridges for culture media. Pure alkaloidal fractions were, thus, analyzed by LC‐ESI‐MS allowing the quantitative evaluation of galanthamine and lycorine from culture extracts. Precursor feeding along with temporary immersion conditions was found to significantly improve the accumulation of both galanthamine and lycorine. The maximal concentrations of galanthamine (0.81 mg/g DW) and lycorine (0.54 mg/g DW) in L. aestivum bulblets were reached, respectively, after 40 days of culture with 0.15 g/L of precursor and after 30 days of culture with 0.3 g/L of precursor. In L. aestivum ‘Gravety Giant’ bulb cultures, 0.3 g/L of precursor was the best condition for both galanthamine (0.6 mg/g DW after 50 days) and lycorine (1.13 mg/g DW after 30 days).     

Improved production of phenylethanoid glycosides by Cistanche deserticola cells cultured in an internal loop airlift bioreactor with sifter riser

An internal loop airlift bioreactor with sifter riser (ILABSR) was composed of a bubble column and a draught-tube rolled with 40-mesh sifter that placed 5 cm above the bottom at the center of the column. A 2 L ILABSR was used for the suspension cultivation of Cistanche deserticola cells and its performance was compared with shake flask culture and a bubble column. Under the optimum culture conditions with the air flowrate of 0.075 m³/h and the inoculation size of 4.7%, about one-fifth cells were attached to the sifter draught-tube. PeG content in these cells was 16.3%, which was 104% higher than that of suspension cells. The production of phenylethanoid glycosides reached 0.85 g/L, which was 102 and 4% higher than those cultured in a 2 L bubble column and shake flasks respectively under their optimal culture conditions.     

Elicitation and in situ adsorption enhanced secondary metabolites production of Tripterygium wilfordii Hook. f. adventitious root fragment liquid cultures in shake flask and a modified bubble column bioreactor

The experiments of elicitation and in situ adsorption were conducted in shake flasks and then tested in a modified bubble column bioreactor for enhancing the productions of three active metabolites in Tripterygium wilfordii Hook. f., triptolide, wilforgine and wilforine. Methyl jasmonate was screened out as the elicitor and the non-ionic polymeric ion-exchange resin of Amberlite^® XAD-7 was used for in situ product removal and protecting the alkaloids from degradation in the medium. In shake flask experiments, 3.55-fold, 49.11-fold, and 10.40-fold of triptolide, wilforgine, and wilforine, respectively, could be recovered from the medium and XAD-7 resin by elicitation and in situ product removal, compared with the control. The modified 10 L bubble column bioreactor had similar productions of the three active metabolites but needed a further optimization of parameters for better growth of adventitious roots.     

Erythropoietin production from CHO cells grown by continuous culture in a fluidized-bed bioreactor.

A Chinese hamster ovary (CHO) cell line that expresses human erythropoietin (huEPO) was in a 2-L Cytopilot fluidized-bed bioreactor with 400 mL macroporous Cytoline-1 microcarriers and a variable perfusion rate of serum-free and protein-free medium for 48 days. The cell density increased to a maximum of 23 x 10(6) cells/mL, beads on day 27. The EPO concentration increased to 600 U/mL during the early part of the culture period (on day 24) and increased further to 980 U/mL following the addition of a higher concentration of glucose and the addition of sodium butyrate. The EPO concentration was significantly higher (at least 2x than that in a controlled stirred-tank bioreactor, in a spinner flask, or in a stationary T-flask culture. The EPO accumulated to a total production of 28,000 kUnits over the whole culture period. The molecular characteristics of EPO with respect to size and pattern of glycosylation did not change with scale up. The pattern of utilization and production of 18 amino acids was similar in the Cytopilot culture to that in a stationary batch culture in a T-flask. The concentration of ammonia was maintained at a low level (< 2 mM) over the entire culture period. The specific rate of consumption of glucose, as well as the specific rates of production of lactate and ammonia, were constant throughout the culture period indicating a consistent metabolic behavior of the cells in the bioreactor. These results indicate the potential of the Cytopilot bioreactor culture system for the continuous production of a recombinant protein over several weeks.     

Elicitation with methyl jasmonate combined with cultivation in the Plantform™ temporary immersion bioreactor highly increases the accumulation of selected centellosides and phenolics in Centella asiatica (L.) Urban shoot culture

Centella asiatica (L.) Urban is an important pharmacopoeial plant used not only in medicine but also in cosmetology. C. asiatica agitated shoot cultures were established to study the influence of ethephon, methyl jasmonate, L ‐phenylalanine (Eth 50 µM, MeJa 50 µM, L‐Phe 2.4 g/L of medium, respectively; seven variants of the supplementation) on the accumulation of secondary metabolites: the main centellosides (asiaticoside and madecassoside) and selected phenolic acids, and flavonoids in the biomass. Microshoots were harvested two and six days after the supplementation. Secondary metabolites were analyzed in methanolic extracts by UPLC‐MS/MS (centellosides) and by HPLC‐DAD (phenolics). In comparison with the reference cultures, the concentrations of individual secondary metabolites increased as follows: centellosides up to 5.6‐fold (asiaticoside), phenolic acids up to 122‐fold (p‐coumaric acid) and flavonoids up to 22.4‐fold (kaempherol). The highest production increase of individual compounds was observed for different variants of supplementation. Variant C (50 µM MeJa), the most optimal for centellosides and flavonoid accumulation, was selected for the experiment with bioreactors. Bioreactor Plantform?, compared to RITA^® system and agitated cultures, appeared to be the most advantageous for secondary metabolites production in C. asiatica shoot cultures. The phenolic acid, flavonoid, centelloside, and total secondary metabolite productivity in Plantform? system is 1.8‐fold, 1.7‐fold, 2.8‐fold, 2.1‐fold, respectively, higher than in MeJa elicitated agitated cultures, and 4.3‐fold, 7.3‐fold, 12.2‐fold, 7.2‐fold, respectively, higher than in control agitated cultures.     

Culture of Scenedesmus sp. LX1 in the modified effluent of a wastewater treatment plant of an electric factory by photo-membrane bioreactor

To investigate the coupled technology for advanced wastewater treatment and microalgal biomass production, a photo-membrane bioreactor was constructed. The microalga Scenedesmus sp. LX1 was cultured in the bioreactor using liquor prepared from the effluent of an electronic device factory. The algal cell growth, nitrate nitrogen removal, orthophosphate phosphorus removal were investigated. When cultured with batch operation, the average specific growth rate was about 0.09 d⁻¹, and low nitrogen (N), phosphorus (P) concentrations in the liquor were achieved. However, under continuous operation with an inflow of 60 L h⁻¹, the average specific growth rate was only 0.02 d⁻¹, and removal rates of 100% for orthophosphate P and 46% for nitrate N were achieved. With the inflow of 120 L h⁻¹, the accumulated metal ions in the bioreactor adversely affected the algal cells. The algal cells were much easier to settle, and the removal efficiency for N and P decreased.     

Flow dynamics within a bioreactor for tissue engineering by residence time distribution analysis combined with fluorescence and magnetic resonance imaging to investigate forced permeability and apparent diffusion coefficient in a perfusion cell culture chamber

This study reveals that residence time distribution (RTD) analysis with pH monitoring after acid bolus injection can be used to globally study the flow dynamics of a perfusion bioreactor, while fluorescence microscopy and magnetic resonance imaging (MRI) were used to locally investigate mass transport within a hydrogel scaffold seeded or not with cells. The bioreactor used in this study is a close‐loop tubular reactor. A dispersion model in one dimension has been used to describe the non‐ideal behavior of the reactor. From open‐loop experiments (single‐cycle analysis), the presence of stagnant zones and back mixing were observed. The impact of the flow rate, the compliance chamber volume and mixing were investigated. Intermediate flows (30, 45, 60, and 90 mL min⁻¹) had no effect over RTD function expressed in reduced time (θ). Lower flow rates (5 and 15 mL min⁻¹) were associated to smaller extent of dispersion. The compliance chamber volume greatly affected the dynamics of the RTD function, while the effects of mixing and flow were small to non‐significant. An empirical equation has been proposed to localize minima of the RTD function and to predict Pe_r. Finally, cells seeded in a gelatin gel at a density of 800,000 cells mL⁻¹ had no effect over the permeability and the apparent diffusion coefficient, as revealed by fluorescent microscopy and MRI experiments. Biotechnol. Bioeng. 2011;108: 2488–2498. © 2011 Wiley Periodicals, Inc.