Defense Response Of Suspension Cultured Taxus Cell In Bubble Column Bioreactor

To investigate the denfense response of suspension cultured Taxus cell in bubble column bioreactor, hydrodynamic stresses in bubble columns with different nozzle diameters were quantitatively analyzed. Physiological responses at the gene transcription, protein enzyme and membrane phospholipids level on sparging bubbles were investigated. Our researches may provide some theory foundations in scaling up of plant cell suspension culture.The hydrodynamic stress fields in bubble columns with different nozzle diameters were studied by laser Doppler anemometer (LDA). It showed that the value of axial normal Reynolds stress was much higher than that of any other Reynolds stress, which had a magnitude from 2.0 to 7.2 N/m2. The location for maximum value of hydrodynamic stress was adjacent to air-liquid interface, then the bubble formation at the sparger, then the main region during bubble rising. Compared the hydrodynamic stresses in bubble columns with different nozzle diameters, the hydrodynamic stresses at the air-liquid interface were increased with small nozzle diameters. It confirmed from a hydrodynamic perspective that gas-liquid interface is the main region where severest damage was happened. Results of hydrodynamic stress fields also showed cells were damaged intensively in bubble column with smaller nozzle sizes.Quantitative RT-PCR was used to evaluate the effect of air sparging on gene transcription levels. In a short-term culture, it led to a decrease of 53.1% and 41.5% for hmgr gene and dxr gene in bubble column with 1.5 mm nozzle diameters, and a decrease of 64.2% and 53.8% in bubble column with 1.0mm nozzle diameters compared with shake flask culture. The ts gene transcription level had an obvious change in the bubble column culture. In a long-term culture, the three genes increased from 6th to 11th day, and declined from day 11th to 26th in bubble columns. The hmgr, dxr and ts gene transcription had an increase of 1.59, 1.51 and 1.47 folds in bubble column with 1.5mm nozzle diameter, while 1.31, 1.31 and 1.26 folds in bubble column with 1.5mm nozzle diameter increase compared with shake flask culture.Several defense responses were generated under the hydrodynamical stresses in bubble columns. Oxidative burst occurred after 8h in bubble columns, followed by NO generation in 11 h. It also indicated PAL and LOX activity reached its maximum value after 16h and 20h. Compared with cells in bioreactor with nozzle diameter of 1.5 mm, ROS, NO, PAL and LOX activity of Taxus are all higher in bioreactor with nozzle diameter of 1.0mm.Metabolic difference of membrane phospholipids in Taxus cuspidate between bubble columns and shake flask culture was analyzed by LC/ESI-MS technology. Principle component analysis was used to distinguish 33 groups according to its phospholipids difference. It indicated that samples between shake flask and bioreactor culture (1.5mm nozzle diameter, 48h) are well clustered according to the PC1 and PC2. The cluster distance of phospholipids sample of bubble column culture for 96h with shake flask culture were farther. MS2 scan were performed to identify the molecular structure of potential biomarker. It indicated that bubble column culture had lead some PA content increased and some PC decreased obviously. Bubble column with smaller nozzle diameter had led the difference of membrane phospholipid more distinctly.