| Plant cell suspension cultivation has proved to be an effective method for offering continuous and stable production for herbal medicine and target compounds. However, in past decades, the researches demonstrate that there still has critical difficulty on engineering amplification technolygy in industrial suspension culture process. Carthamus tinctorius L. is one of the most commonly used Chinese herbal medicines, which was used to prevent and treat cardiac disease in clinical practice. With the quickly increasing market demand, suspension callus cultures have become one of the most potential methods for C. tinctorius L. production. However, the industrial scale-up in stirred tank bioreactors on C. tinctorius L. cells cultivation, fragile and slow-growing effects have always been a serious problem. The hydrodynamic shear stresses generated from the turbulent flow were proved to be the pivotal reasons on industrial C. tinctorius L. cultivation. However, a comprehensive connection between shear and plant cell metabolism is still not well documented. Therefore, the aim of our research is to develop a methodology to combined CFD codes with biological models (scale down model) to make precise scale-up predictions.Firstly, the platform on monitoring the basic physiological characteristics of C. tinctorius L. through online and offline were established. The physiological parameter as oxygen uptake rate (OUR), carbon dioxide evolution rate (CER) and capacitance measurements could be used to represent the characteristics of C. tinctorius L. cell growth. Moreover, this study provided an effective online monitoring methodology for the aggregate size distribution in the suspension culture with viable biomass sensor. The results demonstrated that the changes of the cell aggregate size could be reflected in the ?-dispersion by the multi-frequency capacitance measurements, which is the first time employed on plant cell aggregate size distribution during culture process. Furthermore, effects of C. tinctorius L. cell growth was described from the angle of the environmental factors and the nutrient composition of the medium. The effects of carbon source, amino acid and plant hormones on cell growth were investigated; and also, the light strength, oxygen supply level, shear force and other factors affect the cell growth of C. tinctorius L. cells were also optimized. The results demonstrated that the balance between oxygen supply and shear environment is the core problem on C. tinctorius L. suspension culture and plant cell reactor design.Computational Fluid Dynamics (CFD) model was applicated to quantitative analysis the flow field. First 3D flow field simulation in stirred bioreactor was proposed and then validated by Particle Image Velocimetry (PIV) experiments. Influence of setuped model and method selection on simulation results was also studied. This research present a new hybrid hexahedral and tetrahedral mesh model, the simulation results agree well with the experimental data of this type of grid, which has the advantage on significant reducing the number of grids and improving calculation efficiency in the calculation of large reactor. Secondly, by comparing the various grid density and structure, four kinds of turbulence model and two kinds of rotating region calculation methods (MRF and SM), we found standard k-? model and SM methods were close to the PIV experimental data and cost less computing resource through weighing the resource consumption and accurate simulation results. Further, using the VOF model, the simulation method of shaking flask reactor is constructed. And the engineering parameters such as energy dissipation rate, shear force and mass transfer coefficient are obtained by the simulation results.In addition, we propose a quantitative method for evaluating the lethal and growth effects of hydrodynamic on C. tinctorius L. cell with computational fluid dynamic (CFD) technology and online capacitance viable cell detection. The effects of long and short term shear force on C. tinctorius L. cells were carried out in 5 L bioreactor and shaken flask under various shear intensities. The changes of physiological metabolism including dynamic vital cell numbers, the specific growth rate, the rate of sugar consumption, the size of the cell and so on were studied. A threshold value was achieved at an average and maximum shear stress of approximately 0.55 Pa (0.06 w/kg) and 4.00 Pa (0.93 w/kg) according to the influence on cell growth. And another threshold value was achieved at an average and maximum shear stress of approximately 1.21 Pa (0.2W/kg) and 10.OPa (4.2W/kg) based on shear death. Furthermore, an Euler-Lagrange approach was achieved for quantitative evaluation of peak shear force/circle time's effects on the damage of C. tinctorius L. cell in stirred tank bioreactor, and this results were effectively verified in a 15L STR bioreactor. A good correlation between cell death kinetics and the parameter of peak shear force/circle time has been observed.Finally, an integration model for the description of shear environment caused biological population dynamics in industrial bioreactors is detailed. This model couples the cell growth effect corresponding to the average shear force, and cell death effect corresponding to the local shear force. Considering the model time scale and the simulation time, when simulating a large bioreactor using an Euler-Euler approach, compartment model for hydrodynamics. This approach dissociates the growth rate from local shear environment leading to a good understanding of the effects of a changing environment on a population. Finally, the integration model was applied to predict an amplification process of a 100L reactor for a batch culture, and then the simulation results were verified by the fermentation process. The results demonstrated that although the compartment model tail to describe the details of the shear zone area, but this approach dissociates the growth rate from local shear condition, which reflects the influence of non-uniformity of flow field on cell growth. It is believed that the concepts of integration of computational fluid dynamics and cell structural dynamic model is a promising way to address the issue of simulating industrial bioreactors. |
PDF Full Text Request