A novel centrifugal impeller bioreactor. II. Oxygen transfer and power consumption

The effects of the impeller configuration, aeration rate, and agitation speed on oxygen transfer coefficient K(L)a were studied in a newly designed centrifugal impeller bioreactor (5-L). The oxygen transfer rates in the novel bioreactor were also compared with those in a cell-lift bioreactor with comparable dimensions. The cell-lift impeller produced much higher surface oxygen transfer rates than the centrifugal one at an agitation speed over 200 rpm. This result was in good agreement with our observation that the cell-lift impeller produced much higher unfavorable turbulence. In addition, the experiments using granulated agar particles as pseudo plant cells indicated that the K(L)a value decreased steadily with an increase in agar particle concentration, and the centrifugal impeller still demonstrated a larger K(L)a than the cell lift up to a high pseudo cell concentration of 19.5 g dry weight (DW)/L (under 150 rpm and 0.20 vvm) or 22.3 g DW/L (under 200 rpm and 0.20 vvm). Furthermore, the correlation between power number and impeller Reynolds number for both the centrifugal and the cell-lift impellers was successfully obtained, which could be used for predicting the power input required by each impeller. From the results obtained, the centrifugal impeller bioreactor is expected to have great potential in its application to shear-sensitive biological systems.     

Azadirachtin production in stirred tank reactors by Azadirachta indica suspension culture

Successful scale-up of Azadirachta indica suspension culture for azadirachtin production was done in stirred tank bioreactor with two different impellers. The kinetics of biomass accumulation, nutrient consumption and azadirachtin production of A. indica cell suspension culture were studied in a stirred tank bioreactor equipped with centrifugal impeller and compared with similar bioreactor with a setric impeller to investigate the role of O₂ transfer efficiency of centrifugal impeller bioreactor on overall culture metabolism. The maximum cell mass for centrifugal impeller bioreactor and stirred tank bioreactor (with setric impeller) were 18.7 and 15.5 g/L (by dry cell weight) and corresponding azadirachtin concentrations were 0.071 and 0.05 g/L, respectively. Glucose and phosphate were identified as the major growth-limiting nutrients during the bioreactor cultivation. The centrifugal impeller bioreactor demonstrated less shearing and improved O₂ transfer than the stirred tank bioreactor equipped with setric impeller with respect to biomass and azadirachtin production.     

Simultaneous saccharification and ethanol fermentation at high corn stover solids loading in a helical stirring bioreactor

The higher ethanol titer inevitably requires higher solids loading during the simultaneous enzymatic saccharification and fermentation (SSF) using lignocellulose as the feedstock. The mixing between the solid lignocellulose and the liquid enzyme is crucially important. In this study, a bioreactor with a novel helical impeller was designed and applied to the SSF operation of the steam explosion pretreated corn stover under different solids loadings and different enzyme dosages. The performances using the helical impeller and the common Rushton impeller were compared and analyzed by measuring rheological properties and the mixing energy consumption. The results showed that the new designed stirring system had better performances in the saccharification yield, ethanol titer, and energy cost than those of the Rushton impeller stirring. The mixing energy consumption under different solids loadings and enzyme dosages during SSF operation were analyzed and compared to the thermal energy in the ethanol produced. A balance for achieving the optimal energy cost between the increased mixing energy cost and the reduced distillation energy cost at the high solids loading should be made. The potentials of the new bioreactor were tested under various SSF conditions for obtaining optimal ethanol yield and titer. Biotechnol. Bioeng. 2010. 105: 718–728. © 2009 Wiley Periodicals, Inc.     

Flow Pattern and Mixing in the Multi‐Turbine Agitated Bioreactor Filled with Poly(γ‐Glutamic Acid) Solution

We investigated the flow pattern and mixing behavior of a poly(γ‐glutamic acid) (γ‐PGA) solution in a bioreactor equipped with two Rushton turbines by simulation and experiment. Computational fluid dynamics (CFD) is used to solve the three‐dimensional hydrodynamics in the bioreactor and to obtain the flow patterns and tracer concentration at every point. The flow circulation patterns by inter‐impeller clearance and viscosity and their effects on overall mixing time were studied. Based on the results we can conclude that the impeller clearance should not be larger than 0.2 D for the efficient mixing under non‐aerated condition when the liquid viscosity is above 20 cp, which corresponds to concentrations of 20 g/L or above for γ‐PGA.     

机械搅拌式动物细胞反应器不同桨型组合的CFD数值模拟与优化

生物反应器已成为哺乳动物细胞生产治疗性抗体药物和疫苗的核心。文中采用CFD数值模拟方法对目前常用的机械搅拌式生物反应器在不同的搅拌形式下的流场进行了分析,获得了5种搅拌桨型组合条件下的速率矢量、持气率、含气率和剪切力分布的特征。通过构建的重组CHO细胞在不同搅拌形式条件下的流加分批培养发现,细胞密度和抗体表达水平与反应器内的最大剪切率直接相关,在FBMI3搅拌形式下细胞密度和抗体表达水平均最高。结果表明该CHO细胞在悬浮培养时对剪切环境比较敏感,且最大剪切力是工业规模放大的关键因素。     

A comprehensive comparison of mixing, mass transfer, Chinese hamster ovary cell growth, and antibody production using Rushton turbine and marine impellers

Large scale production of monoclonal antibodies has been accomplished using bioreactors with different length to diameter ratios, and diverse impeller and sparger designs. The differences in these physical attributes often result in dissimilar mass transfer, mechanical stresses due to turbulence and mixing inside the bioreactor that may lead to disparities in cell growth and antibody production. A rational analysis of impeller design parameters on cell growth, protein expression levels and subsequent antibody production is needed to understand such differences. The purpose of this study was to examine the impact of Rushton turbine and marine impeller designs on Chinese hamster ovary (CHO) cell growth and metabolism, and antibody production and quality. Experiments to evaluate mass transfer and mixing characteristics were conducted to determine if the nutrient requirements of the culture would be met. The analysis of mixing times indicated significant differences between marine and Rushton turbine impellers at the same power input per unit volume of liquid (P/V). However, no significant differences were observed between the two impellers at constant P/V with respect to oxygen and carbon dioxide mass transfer properties. Experiments were conducted with CHO cells to determine the impact of different flow patterns arising from the use of different impellers on cell growth, metabolism and antibody production. The analysis of cell culture data did not indicate any significant differences in any of the measured or calculated variables between marine and Rushton turbine impellers. More importantly, this study was able to demonstrate that the quality of the antibody was not altered with a change in the impeller geometry.     

Technological problems in cultivation of plant cells at high density. Reprinted from Biotechnology and Bioengineering, Vol. XXII, No. 6, Pages 1203-1218 (1981)

Using Cudrania tricuspidata cells as model plant cells which have high sensitivity to hydrodynamic stress, technological problems in the cultivation of the plant cells at high density were investigated. Using "shake" flasks on a reciprocal shaker and Erlenmeyer flasks on a rotary shaker and with a high supply of oxygen in order to obtain high cell densities in shaken cultures, particle breakdown and damage to the largest cell aggregate group (above 1981 microm in diameter) occurred and normal cell growth became impeded. The mass-transfer coefficient (K) for a model solid-liquid system (beta-naphthol particles and water) in place of a system of plant cells and a liquid medium was proposed as an intensity index of hydrodynamic stress effects on plant cells in suspension cultures under various conditions in the bioreactor systems. Normal cell growth was obtained under culture conditions for K values less than about 4.4 x 10(-3) cm/sec. The characteristics of various bioreactors used until now were investigated by considering the three main technological factors (capacity of oxygen supply, intensity of hydrodynamic stress effects on plant cells, and intensity of culture broth mixing and air-bubble dispersion). The most suitable bioreactor for culturing plant cells at high density was a jar fermentor with a modified paddle-type impeller (J-M). The yield of cell mass in the 10-liter J-M (working volume 5 liter) was about 30 g dry weight per liter of medium.     

Development of a helical-ribbon impeller bioreactor for high-density plant cell suspension culture

A double helical-ribbon impeller (HRI) bioreactor with a 11-L working volume was developed to grow high-density Catharanthus roseus cell suspensions. The rheological behavior of this suspension was found to be shear-thinning for concentrations higher than 12 to 15 g DW . L(-1). A granulated agar suspension of similar rheological properties was used as a model fluid for these suspensions. Mixing studies revealed that surface baffling and bottom profiling of the bioreactor and impeller speeds of 60 to 150 rpm ensured uniform mixing of suspensions. The HRI power requirement was found to increase singnificantly for agar suspensions higher than 13 g DW . L(-1), in conjunction with the effective viscosity increase. Oxygen transfer studies showed high apparent surface oxygen transfer coefficients (k(L)a approximately 4 to 45 h(-1)) from agar suspensions of 30 g DW . L(-1) to water and for mixing speeds ranging from 120 to 150 rpm. These high surface k(I)a values were ascribed to the flow pattern of this bioreactor configuration combined with surface bubble generation and entrainment in the liquid phase caused by the presence of the surface baffles. High-density C. roseus cell suspension cultures were successfully grown in this bioreactor without gas sparging. Up to 70% oxygen enrichment of the head space was required to ensure sufficient oxygen supply to the cultures so that dissolved oxygen concentration would remain above the critical level (>/=10% air saturation). The best mixing speed was 120 rpm. These cultures grew at the same rate ( approximately 0.4 d(-1)) and attained the same high biomass concentrations ( approximately 25 to 27 g DW . L(-1), 450 to 500 g filtered wet biomass . L(-1), and 92% to 100% settled wet biomass volume) as shake flask cultures. The scale-up potential of this bioreactor configuration is discussed.     

A novel impeller configuration to improve fungal physiology performance and energy conservation for cephalosporin C production

Effects of impeller configuration on fungal physiology and cephalosporin C production were investigated by an industrial strain Acremonium chrysogenum in a 12m(3) bioreactor equipped with conventional and novel impeller configuration, respectively. The cell growth and oxygen uptake rate (OUR) profiles were little affected by the impeller configurations. However, differing impeller combinations significantly affected the morphology, which in turn influenced cephalosporin C production. Under the novel impeller configuration, the production of cephalosporin C was 10% higher and an excessive amount of dispersed arthrospores was also observed. Computational fluid dynamics (CFD) simulation further revealed that poor mass and energy exchange as well as inhomogeneous environment existed in the bioreactor equipped with conventional impeller configuration. For equivalent power dissipation, the volume oxygen transfer coefficient (K(L)a) could be enhanced by 15% compared with that of conventional impeller configuration. Power consumption was dramatically decreased by 25% by using novel impeller configuration.     

Comparison of O transfer characteristics between a new centrifugal impeller and a flat-bladed turbine impeller

The efficiency of O transfer by a novel centrifugal impeller was higher than that of a conventional flat-bladed turbine impeller at an agitation speed lower than 300 rpm. In addition, at the same agitation speed (200 and 300 rpm), the centrifugal impeller possessed smaller shear stress than the flat-bladed turbine impeller as evaluated by the changes in size distribution of granulated agar particles which were sheared with those two types of impeller.     

Scale-up of stirring as foam disruption (SAFD) to industrial scale

Foam disruption by agitation—the stirring as foam disruption (SAFD) technique—was scaled up to pilot and production scale using Rushton turbines and an up-pumping hydrofoil impeller, the Scaba 3SHP1. The dominating mechanism behind SAFD—foam entrainment—was also demonstrated at production scale. The mechanistic model for SAFD defines a fictitious liquid velocity generated by the (upper) impeller near the dispersion surface, which is correlated with complete foam disruption. This model proved to be scalable, thus enabling the model to be used for the design of SAFD applications. Axial upward pumping impellers appeared to be more effective with respect to SAFD than Rushton turbines, as demonstrated by retrofitting a 12,000 l bioreactor, i.e. the triple Rushton configuration was compared with a mixed impeller configuration from Scaba with a 20% lower ungassed power draw. The retrofitted impeller configuration allowed 10% more broth without risking excessive foaming. In this way a substantial increase in the volumetric productivity of the bioreactor was achieved. Design recommendations for the application of SAFD are given in this paper. Using these recommendations for the design of a 30,000 l scale bioreactor, almost foamless Escherichia coli fermentations were realised. Electronic Publication     

Effect of Impeller Clearance on Flow Structure and Mixing in Bioreactor with Two‐Stage Impellers

The effect of impeller clearance on flow structure and mixing time was simulated using a commercial software package CFX 4.3 and was measured experimentally. The mixing time calculated by simulation exhibited good agreement with the experimental data. The trend of forming independent flow compartments by each impeller became stronger as the clearance between two impellers increased. The homogeneity in the bioreactor was affected mainly by flow exchange between the compartments by each impeller. The most efficient mixing occurred when the impeller clearance was in the range of 0.2–0.4 vessel diameter.     

Homogeneous batch cultures of<Emphasis Type="Italic"> Aspergillus oryzae</Emphasis> by elimination of wall growth in the Variomixing bioreactor

A novel principle for mixing and aeration in stirred bioreactors, named Variomixing, was developed. Four baffles are rotated intermittently at a rotational speed slower or similar to the speed of a centrally placed axial flow impeller. Rotational speeds of the baffles and impeller of 5–10 and 500–600 rpm, respectively, results in the highly turbulent flow regime characteristic of conventional bioreactors with high mixing and mass transfer capacities. Stagnant zones around crevices and crannies in which wall growth may commence are avoided since the baffles are never completely at rest. Increasing the rotational speed of the baffles (5 s every 5 min), so that it follows the speed of the impeller (500–600 rpm), cancels the effect of the baffles and a deep vortex and high peripheral liquid flow rates at the reactor wall develop. The vortex ensures that also the head-space of the reactor wall is flushed and any deposits removed. The filamentous fungus Aspergillus oryzae has been grown in batch cultures in the Variomixing bioreactor. Compared to conventional laboratory-scale bioreactors, in which more than 30% of all biomass was found attached to walls, less than 2% of the total A. oryzae biomass was found on the walls in the Variomixing bioreactor.     

Hydrodynamic and mass transfer characteristics of three-phase gaslift bioreactor systems.

This review focuses on the hydrodynamic and mass transfer characteristics of various three-phase, gaslift fluidized bioreactors. The factors affecting the mixing and volumetric mass transfer coefficient (k(L)a), such as liquid properties, solid particle properties, liquid circulation velocity, superficial gas velocity, bioreactor geometry, are reviewed and discussed. Measurement methods, modeling and empirical correlations are reviewed and compared. To the authors' knowledge, there is no 'generalized' correlation to calculate the volumetric mass transfer coefficient, instead, only 'type-specific' correlations are available in the literature. This is due to the difficulty in modeling the gaslift bioreactor, caused by the variation in geometry, fluid dynamics, and phase interactions. The most important design parameters reported in the literature are: gas hold-up, liquid circulation velocity, 'true' superficial gas velocity, mixing, shear rate, aeration rate and volumetric mass transfer coefficient, k(L)a.     

Characterization and application of a miniature 10 mL stirred-tank bioreactor, showing scale-down equivalence with a conventional 7 L reactor

The aim of this study was to characterize the engineering environment of an instrumented 10 mL miniature stirred-tank bioreactor and evaluate its potential as a scale-down device for microbial fermentation processes. Miniature bioreactors such as the one detailed in this work have been developed by several research groups and companies and seek to address the current bottleneck at the screening stage of bioprocess development. The miniature bioreactor was characterized in terms of overall volumetric oxygen transfer coefficient and mixing time over a wide range of impeller speeds. Power input to the miniature bioreactor was directly measured, and from this the power number of each impeller was calculated and specific power input estimated, allowing the performance of the miniature bioreactor to be directly compared with that of a conventional 7 L bioreactor. The capability of the miniature bioreactor to carry out microbial fermentations was also investigated. Replicate batch fermentations of Escherichia coli DH5alpha producing plasmid DNA were performed at equal specific power input, under fully aerobic and oxygen-limiting conditions. The results showed a high degree of equivalence between the two scales with regard to growth and product kinetics. This was underlined by the equal maximum specific growth rate and equal specific DNA product yield on biomass obtained at the two scales of operation, demonstrating the feasibility of scaling down to 10 mL on the basis of equivalent specific power input.     

Macromixing hydrodynamic study in draft-tube airlift reactors using electrical resistance tomography

The present study summarizes results of mixing characteristics in a draft tube airlift bioreactor using ERT. This technique offers the possibility for noninvasive and nonintrusive visualization of flow fields in the bioreactor and has rarely been utilized previously to analyze operating parameters and mixing characteristics in this type of bioreactors. Several operating parameters and geometric characteristics were examined. In general, results showed that the increase in superficial gas velocity corresponds to an increase in energy applied and thus, to a decrease in mixing time. This generally corresponded to an increase in liquid circulation velocity and shear rate values. Bottom clearances and draft tube diameters affected flow resistance and frictional losses. The influence of sparger configurations on mixing time and liquid circulation velocity was significant due to their effect on gas distribution. However, the effect of sparger configuration on shear rate was not significant, with 20% reduction in shear rates using the cross-shaped sparger. Fluid viscosity showed a marked influence on both mixing times and circulation velocity especially in the coalescing media of sugar and xanthan gum (XG) solutions. Results from this work will help to develop a clear pattern for operation and mixing that can help to improve several industrial processes, especially the ones related to emerging fields of technology such as the biotechnology industry.     

Technological problems in cultivation of plant cells at high density

Using Cudrania tricuspidata cells as model plant cells which have high sensitivity to hydrodynamic stress, technology problems in the cultivation of the plant cells at high density were investigated. Using “shake” flasks on a reciprocal shaker and Erlenmeyer flasks on a rotary shaker and with a high supply of oxygen on order to obtain high cell densities in shaken cultures, particles breakdown and damage to the largest cell aggregate group (above 1981 μm in diameter) occurred and normal cell growth became impeded. The mass-transfer coefficient (K)for a model solid–liquid system (β-naphthol particles and water) in place of a system of plant cells and a liquid medium was proposed as an intensity index of hydrodynamic stress effects on plant cells in subsequent cultures under various conditions in the bioreactor systems. Normal cell growth was obtained under culture conditions for K values less than about 4.4 × 10^?3 cm/sec. The characteristics of various bioreactors used until now were investigated by considering the three main technological factors (capacity of oxygen supply, intensity of hydrodynamic stress effects on plant cells, and intensity of culture broth mixing and air-bubble desperation). The most suitable bioreactor for culturing plant cells at high density was ajar fermentor with a modified paddle-type impeller (J-M). The yield of cell mass in the 10-liter J-M (working volume 5 liter) was about 30 g dry weight per liter of medium.     

Evaluation of mixing effect and shear stress of different impeller combinations on nemadectin fermentation

Mass transfer and shear force have significant effects on nemadectin production by Streptomyces cyaneogriseus ssp. noncyanogenus. They are always the conflict-ridden problems in nemadectin fermentation process. In this study, the flow field characteristics under different impeller combinations were quantitatively evaluated in 5 L stirred-tank bioreactor through the laser particle image velocimetry (PIV) system. Results demonstrated that the radial-axial impeller combinations with the time average velocity at 0.38-0.54 U_tip, the turbulent kinetic energy dissipation rate at 6.4–10.6 ε/N³D², and the shear stress rate was 40-150 s⁻¹, were more conductive to cell growth, nemadectin biosynthesis, cell’s activity, respiratory metabolism than other combinations. The highest nemadectin yield was evaluated up to 1543.3 ± 18.5 μg/mL, which was 31.68 % higher than that of the radial flow impeller combinations. This study provided the important guideline for the selection impeller combinations’ on large-scale nemadectin production.     

Scale-up analysis of geometrically dissimilar single-use bioreactors

Cell culture scale-up is a challenging task due to the simultaneous change of multiple hydrodynamic process characteristics and their different dependencies on the bioreactor size as well as variation in the requirements of individual cell lines. Conventionally, the volumetric power input is the most common parameter to select the impeller speed for scale-up, however, it is well reported that this approach fails when there are huge differences in bioreactor scales. In this study, different scale-up criteria are evaluated. At first, different hydrodynamic characteristics are assessed using computational fluid dynamics data for four single-use bioreactors, the Mobius® CellReady 3 L, the Xcellerex™ XDR-10, the Xcellerex™ XDR-200, and the Xcellerex™ XDR-2000. On the basis of this numerical data, several potential scale-up criteria such as volumetric power input, impeller tip speed, mixing time, maximum hydrodynamic stress, and average strain rate in the impeller zone are evaluated. Out of all these criteria, the latter is found to be most appropriate, and the successful scale-up from 3 to 10 L bioreactor and to 200 L bioreactor is confirmed with cell culture experiments using Chinese Hamster Ovary cell cultivation.     

Mixing studies in bioreactors

Blend times and power consumptions were determined for different arrangements of two equal diameter impellers, a high efficiency A310 and a “Dumbo Ear” impeller with three large, “elephant ear” blades designed for low shear agitation. A 9 l round-bottomed, unbaffled bioreactor was used in these studies. Blend times were taken as the time for the disappearance of the pink color of a basic solution of phenolphthalein on neutralization by excess acid, and the power consumption was obtained from torque measurements. The mixing results show that the Dumbo Ear impeller gives shorter blend times than the A310?at equal rotational speeds for most of the conditions studied. As expected, the Dumbo Ear impeller consumes more power than the A310?at the same rotational speed, due to its large area blades. However, the Dumbo Ear impeller also gives shorter blend times than the A310?at equal power consumptions.