Liquid Mixing and Oxygen Supply

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Biotechnology Journal
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Research Article Insights into Large Scale Cell Culture Reactors: I. Liquid Mixing and Oxygen Supply
Christian Sieblist1,2, Marco Jenzsch2, Michael Pohlscheidt2, Andreas Lbbert1 Center for Bioprocess Engineering, Martin-Luther-University Halle-Wittenberg, Halle, Germany 2 Pharmaceutical Biotech Production & Development, Roche Diagnostics GmbH, Penzberg, Germany
Keywords:
cell culture, oxygen mass transfer, mixing, large scale bioreactors
Corresponding Author:
Professor Andreas Lbbert Center for Bioprocess Engineering Institute of Biochemistry and Biotechnology Martin-Luther-University Halle-Wittenberg Kurt-Mothes-Str. 3 / 320.2 D-06120 Halle/Saale Germany Tel.: +49 345 5525 942 Fax: +49 345 5527 260 E-Mail: Andreas.Luebbert@biochemtech.uni-halle.de
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an Accepted Article, doi: 10.1002/biot.201000408. Submitted: Revised: Accepted: 03-Mar-2011 21-Jul-2011 26-Jul-2011
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Po
Abbreviations A315 CCD CFD cGMP CHO CIP CO2 CTA FDA LDA LES O2 PB pH rpm RT vvm
Lightnin's hydrofoil impeller A315 Charge Coupled Device Computational Fluid Dynamics current Good Manufacturing Practice Chinese Hamster Ovary Cells Clean In Place carbon dioxide Constant Temperature Anemometry Food and Drug Administration Laser Doppler Anemometry Large Eddy Simulation oxygen pitched blade turbine measure of acidity or basicity of an aqueous solution round per minute [1/min] Rushton turbine (volumetric flow rate/minute) / volume of broth, minute European Medicines Agency CFD calculation model for turbulent energy dissipation turbulent Power or Newton number
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Abstract
In the pharmaceutical industry it is state of the art to produce recombinant proteins and antibodies with animal cell cultures using bioreactors with volumes of up to 20 m. Recent guidelines and position papers for the industry by FDA and EMA stress the necessity of mechanistic insights into large scale bioreactors. A detailed mechanistic view of their practically relevant subsystems is required as well as their mutual interactions, i.e. mixing or homogenization of the culture broth and sufficient mass and heat transfer. In large scale bioreactors for animal cell cultures different agitation systems are employed.
Here we discuss details of the flows induced in stirred tank reactors relevant for animal cell cultures Also solutions of the governing fluid dynamic equations obtained with the so-called computational fluid dynamics (CFD) are given. Experimental data obtained with improved measurement techniques are shown. The results are compared to previous studies and it is found that they support current hypotheses or models. Progress in improving insights requires continuous interactions between more accurate measurements and physical models. The paper aims at promoting the basic mechanistic understanding of transport phenomena that are crucial for large-scale animal cell culture reactors.
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1 Introduction
In general, the task of bioreactors is providing the environment to the cells that capacitates them to an optimal performance and, particularly, to a reproducible product quality. In most industrial large-scale production processes for recombinant therapeutic proteins, animal cells are employed. The cells are suspended in continuous liquid culture media; since such submerged cultures most easily allow tightly controlling their environmental conditions. As the cells metabolically respond to changes in their chemical environment, the goal must be to provide optimal conditions for nearly all cells in the vessel, i.e. one is interested in homogeneous conditions within the culture. Bioreactors are employed to provide homogeneous conditions not only with respect to the chemical/biochemical but also to the physical environment of the cells in a culture. The general literature on bioreactors for animal cell cultures was recently reviewed in depth by many specialists [1, 2, 3, 4, 5, 6]. Therefore, here, the literature is only briefly covered in order to set this paper into context. Achieving homogeneity by means of agitation of the cultures with high power inputs has not been considered a solution of that problem because of the concern for cell damaging, since animal cells do not possess a rigid protecting cell wall [7]. Hence, substrate gradients that cannot be avoided in the large-scale bioreactors, lower cell yield and increase byproduct formation [8]. In large cell culture reactors, additionally, the product quality might be affected. Serrato et al. [9] described a case where heterogeneous conditions in the dissolved oxygen affect Nglycosylation more sensitively than the productivity with respect to a monoclonal antibody formation in hybridoma cultures. Godoy-Silva et al. [6] showed that the physiological responses of CHO cells to repetitive hydrodynamic stress, which these cells experience during their stay in large scale reactors, probably do not affect productivity but when there is an influence at all, the glycosylation pattern of the products is impaired.
The difficulty in achieving homogeneity in the often applied fedbatch processes mostly arises from the fact that substrates and auxiliary material must be fed to the reactor, e.g., to supply the cells with nutrients or to compensate for pH changes due to organic acids released by the cells. In order to keep the cultivations under close control, the added substances must immediately, i.e. as fast as possible be completely mixed across the entire culture. This is a transport problem, which is approached by inducing appropriate fluid flows within the continuous liquid phase. This is particularly important for oxygen which is only sparsely soluble and is quickly consumed by the cells.
Oxygen mass transfer is all the way a key task of bioreactors for aerobic cultures. In large reactors, oxygen is usually supplied by dispersing air at the bottom of the reactor. The bubbles rise in the liquid and burst when they leave the culture at its top surface. This process is accompanied by extremely high local shear stresses that are known to be lethally damaging to animal cells. To cope protecting agents such as the block polymer Pluronic F68 were added to the culture [10, 11, 12, 13]. In order to keep these forces lower, larger bubbles are preferred. However, at the same gas throughput, larger bubbles reduce the specific interfacial area for the oxygen mass transfer and thus the net transfer rate. Hence, there are conflicting design objectives. In animal cell cultures we are additionally faced with the problem to withdraw the carbon dioxide that is produced by the cells at roughly the same molar rate as oxygen is consumed. In large bioreactors for animal cell cultures this appears to be a problem as it was observed that in these reactors CO2 accumulates up to concentrations where cell growth and product formation become severely reduced (e.g. Gray et al. [14]).
Industrial production of recombinant protein is nearly entirely performed with aerated stirred tank reactors where the gas is dispersed with so-called gas spargers [15]. A clear advantage of this reactor type is that it provides some operational flexibility as it is possible to influence homogenization and mass transfer by means of three adjustable variables the stirrer speed, the aeration rate and the gas mix. Hence we mainly restrict the discussion here to aerated stirred tank bioreactors. In this paper we aim in providing insights into the physical interrelationships from the mechanistic point of view. This is a key for understanding the main production equipment and it is quite embarrassing that the authorities FDA/EMA [16] felt that they must stress manufacturers to put more emphasis on a knowledge-based approach. Mixing is significantly influenced by the aeration and on the other hand the aeration influences mixing and mass transfer in many respects. The interrelationships are used to explain the different approaches of industrial companies.
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There are several excellent reviews on large scale bioreactors from prominent authors. Here we do not aim in a comprehensive review but alternatively, in a discussion of the physical details of the flow in these bioreactors and their mutual interactions. Instead of traditionally expressing experimental results in terms of engineering correlations we look for physical models for the relevant phenomena and try to explain them with experimental results usually not appearing in the mentioned reviews or with data especially measured for that purpose. As the matter is rather complex we divide the discussion into two parts. In a first we mainly consider mixing and oxygen transfer (this paper) and in the second we focus on the motion of the gas phase and the carbon dioxide stripping problem [17].
2 Materials and methods

For characterization experiments acrylic glass vessels with 200 [L] and 400 [L] working volume were used. All vessels were equipped with four baffles (w/T = 0.1), a dished boiler end and with 1- or 3- stage impeller systems (D/T = 0.33). These model vessels were geometrically similar to those used in production.
To determine the power input torque transducers from ETH-Messtechnik, Geschwend with a measurement range of 2, 5 and 30 [Nm] were used. In addition these sensors were equipped with a rotation speed measurement system. All transducers are mounted with two bellow couplings between drive and impeller shaft to prevent damaging due to radial and axial forces. To record continuously torque and impeller speed, a GMV2 evaluation device from ETH Messtechnik was used. The total power input can be calculated with
Ptotal = Pstirrer + Pgas + Pfriction
(1)
The stirrer an the friction part has been measured with the torque transducers. The power input by gas has been calculated from gas pressure in the sparger. For mixing experiments decolorisation method with the iod-iodine system was applied. This method is described in detail by Henzler 1998 [18]. According to his explanation, a degree of mixing of 95% was chosen to assure a sufficient uniformity in the stirred vessel. The same degree of mixing was used for the CFD simulations and for physical mixing experiments in the production vessels via conductivity probes. The kLa measurements were based on the well-known gassing-in / gassing out method [19]. As measuring system we used the Presens Fibox device in combination with oxygen sensor spots for the experimental vessels. In production vessels the kLa measurements were made with standard dO-probes or with the Presens oxygen probe for in-line measurements.
3 Results and discussion

3.1 Demand on the two-phase flow in stirred tank bioreactors: Turbulent flows
The essential task of a bioreactor is to act as a transport system for substrates and metabolic products within the culture. This transport is accomplished by fluid flows. The easiest way of achieving homogenization in the culture liquid is to induce turbulent flows which guarantee spatial dispersion of adjacent fluid elements disproportionately high with respect to time. However, such turbulent flows are highly dissipative and must thus be supplied with considerable power inputs. In stirred tank bioreactors, turbulent flows are mainly supplied with energy by means of mechanical agitators but also by aeration. The main mechanism is that the impellers generate flows on the reactor scale that are inherently unstable and decay into a series of smaller eddies. Importantly, the size of turbulent fluid elements has a lower limit where the tangential fluid velocity of the eddies roughly the same as the velocity of the radial dispersion of the matter making up the eddy due to molecular diffusion, so that any eddy motion gets lost. Based on this idea the size of the smallest eddies was estimated by Kolmogorov
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=
v
3
[20]. He showed that depends on the rate energy introduced per unit liquid mass by the agitator on the large scale and the kinematic viscosity
1 4
(2)
For instance, an energy input of 0.1 [W/kg], which is currently considered high for animal cell cultures, leads to an estimate for the smallest eddy diameter of about 0.06 [mm]. As the cells, even in animal cell cultures, are smaller as nearly all eddies in the turbulent flows, they are essentially carried along with the liquid, so that it is sufficiently accurate to consider the fluid as a two-phase gas-liquid system.
As opposed to microbial fermentations, a major concern in cell culture technology is whether or not animal cells, which are not protected by rigid cell walls, are destroyed by turbulent eddies. This question is nearly answered with the discussion of the motion of the cells within turbulent liquid flows. With respect to stirred tank reactors many practitioners in industry argument that cell damaging may then be induced by so-called high shear impellers such as Rushton turbines where at the impeller tips high local shear rates (not necessarily turbulent eddies) appear. However, even the highest shear rates that can appear at Rushton impeller tips in production-scale bioreactors are far less than those which are known to be lethal to the usually used cells. Experimentally this was shown by a several researchers most recently in a notable work performed in Jeff Chalmers group at Ohio State University [6]. They showed that their CHO cells withstand power inputs at least up to 6000 [W/kg]. The only impact observed was that the glycosylation pattern of the products gets influenced at higher power inputs. At power dissipation rates in access of 300 [W/kg], which is still much higher than the power densities appearing in commercial cell culture reactors, Godoy-Silva et al. [6] found that the product quality, not the cells as such, becomes influenced. Even if this result cannot be transferred to all other cell cultures, it shows that shear damaging was mostly overestimated in recent decades. This conclusion was also presented in different papers by Nienow (e.g. [3, 4]). In engineering one generally tries to formulate process knowledge by means of process models and validates them by comparing the solutions with experimental result. Currently, much effort is put into fluid flow modeling under the catchword computational fluid dynamics (CFD). This is an extremely difficult task as the computational power required for a computation with a full resolution of all flow details is impossible. This can easily be estimated from Eq. (2). We immediately see that the full resolution of the eddy motion within the computations would require at least L/ grid points, i.e. more than 104 per meter in each of the three dimensions. This is completely out of range of currently available computers. However, we are now becoming able to compute the main properties of the fluid flow patterns which are so essential to mixing in bioreactors. These computations are performed using the large eddy simulation (LES)-procedure, which eliminates the small scale solutions from the Navier-Stokes-Equation-System by replacing the effects on the smaller scales by a so-called turbulence model, such as the k--model. In this way, computations with numerical grids that we can cope with on todays computers become possible [21, 22]. Consequently all fluid motions on these small scales cannot be expected in the computational results. Only large eddy motions can be resolved in these investigations. The fluid flow models are still three-dimensional and highly nonlinear. As such their results depict chaotic solutions in the physical sense, exactly what we like to have in the real flows: turbulence. In these computations, however, as well as in the measurements performed in order to provide the data that are necessary to validate the computations, we must perform some long time averaging, i.e., we must remove the highly fluctuating part before we can compare measurements with computational results. Although one averages away an important part of the physics, particularly the chaotic fluid motion, which is so important to fluid mixing, this is the only way to compare CFD results with experimentally obtained measurements. A typical example of a mean ungassed fluid flow pattern is depicted in figure 1. Figure 1 shows examples of production reactors equipped with conventional Rushton turbines or pitched blade turbines [23]. In comparison CFD results for single phase applications generally associated with such impeller configurations are presented as well. These simulations depict long-time averages of the fluid motions which clearly
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indicate flow compartments. For Rushton turbines a toroid-like flow field appears around each impeller. For pitched blade turbines it comes to a circulation flow through the whole reactor. A first result from basic CFD simulations for Rushton turbines shown in figure 1 is that in the case that the total energy input rate per mass mean is 0.1 [W/kg], which is much higher than the rates usually applied in production reactors, the ratio of max/mean is 70, which leads to max= 7 [W/kg].
The mechanical power transferred into an unaerated liquid by means of an agitator is often related to the third power of the stirrer speed N and, importantly, to the fifth of the impeller diameter D:
P = Po N 3 D 5
(3)
The constant Po, referred to as turbulent Power or Newton number, contains the impeller specific properties. Our current CFD programs are now able to estimate these power numbers quite well. For a Rushton impeller with diameter D=0.33T we obtained from CFD a Po=4.96, which is quite close to the value obtained experimentally. This says that the ungassed power input to the reactors can be simulated fairly accurately.
All large-scale production reactors of this type known to the authors are equipped with 2 to 4 baffles (an example can be seen in the photograph), in order to prevent the development of swirling flows. If the baffles would be withdrawn the impellers would generate flows in which much of the energy put into the fluid motion would be present in a global circulatory motion around the impeller shaft and gets lost for the turbulent motion needed for good mixing [25]. Additionally, the central vortex developing at the culture surface may leads to uncontrolled dispersion of the gas in the head space into the culture broth. Tank designs where eccentrically introduced impeller shafts are used instead of baffles is a solution only where baffles are inconvenient such as in disposable reactors mostly equipped with a single impeller. However, in cylindrical mixing vessels, such constructions are known to need roughly twice the power input to obtain the same mixing time as the constructions with concentric shafts a fact well known since many decades in classical chemical engineering [26]. But not all stirred single-use bioreactors have such eccentric shaft with a single impeller. E.g. the Satourious Biostat CultiBag STR. There existing some other single use systems, available from different suppliers, which have much more different mixing systems which isnt direct comparable to classical impellers e.g. Bayer: BaySHAKE. This system is also available up to 2 [m] working volume. Impellers and baffles are thus necessary and must be considered a pair that works together for sufficient mixing. Myers et al. [26] showed that the power numbers of the various impellers are sensitively dependent on the baffles. Merely an increase of the baffle width leads to an enhancement of the power input to the tank at the same stirrer speed [27]. In microbial bioreactors, this was already used to increase the mass transfer.
The motion of the gas phase through the culture influences the flow in a stirred tank reactor at a given stirrer speed. As low pressure areas develop behind the impeller blades, gas bubbles are attracted to that region, coalesce and form so-called gas cavities behind the blades. As the continuous liquid must flow around these cavities, it effectively sees voluminous impeller blades depicting a reduced flow resistance. Hence, at a fixed stirrer speed, the power draw by the impellers becomes reduced. Cavity formation is not restricted to Rushton impellers. All impeller types produce pressure gradients and thus form more or less voluminous cavities. The cavities can be made visible with CCD camera attached to the impeller shaft. In the examples are shown in figure 2, the formation of a large cavity upon starting the aeration in the stirred tank reactor is shown, i.e. they cover very small aeration rates up to the maximal aeration rates used in animal cell cultures. In all cases cavity formation started at two places at the upper and the lower edge of the impeller blade as can be seen in the figure 2.
The cavity formation and dynamics can also be understood with CFD [21]. A typical result from such simulation, made by A. Bakker, ANSYS Inc., is shown in figure 2 (part B). That picture clearly shows that the vorticity structure compares well with the cavities that can be observed during the visual inspection with a CCD camera shown in figure 2 (part A). Cavity formation is not restricted to Rushton turbines. Vortices that attract bubbles to form gas cavities also appear even behind large impeller blades of axial transporting impellers such as the so-called elephant ears discussed with respect to their use in animal cell culture reactors (e.g. Meier [28]). Figure 3 depicts three photographs showing that
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the cavities develop in extended form at the upper rear edge of the impeller blades. As these cavities are very long, they may become unstable. This means that they disconnect from the impeller blade and disengage as a large bubble from the culture. At the same time the flow resistance of the impeller blade becomes larger until a new cavity develops. This leads to torque instabilities and some rocking of the reactor. For practical work, the reduction in the power input, at a given stirrer speed, is of high importance as the power that is introduced into the culture determines nearly all properties of the flow, from mixing to mass and heat transfer rates. The effect leading to the results depicted in figure 4 can immediately be understood in the following way: At a given stirrer speed, an increasing gas load leads to an increasing size of the cavities and, hence, to a decreasing power transfer into the liquid motion. At a given aeration rate, an increasing stirrer speed leads to an increasing liquid recirculation around the impellers. With the higher circulation velocities around the impellers, more gas is drawn back to the impeller hub thus feeding the gas cavities with the same effect as with increased gas flow rates. This mechanism as was conceptually found long years ago [29, 30].
More detailed experimental investigations of the fluid velocity patterns, particularly the fluid recirculations around the impellers in the bioreactors, require local measurement techniques. For single-phase flows this is possible with Laser Doppler Anemometry (LDA) or Constant Temperature Anemometry (CTA). In two-phase flow environments, however, both techniques are restricted to very low gas holdups, and even then their measurement results are heavily distorted by the bubbles, i.e., the dispersed gas phase. All these local probing techniques depict measurement volumes smaller than the bubble diameters (1-8 [mm]). Hence, they deliver invalid liquid velocity signals when the measurement volume enters a bubble, when the sensors happen to be insides a bubble or when it passes back into the liquid phase. Even if these segments are cut off the signal, the rest contains significant distortions in form of signal jumps. Bubble velocity distributions have been measured with conductivity probes and Ultrasound Doppler techniques [31] which depict larger measurement volumes. Bubbles are known to be excellent reflectors for ultrasound. More than 99% of an ultrasound beam of some [MHz] sound frequency is reflected at their surfaces. Frequency and intensity of the reflected ultrasound carry important information on the properties of the reflecting bubbles. The bubble velocities are obtained from the Doppler shift of the ultrasound frequency reflected from a moving bubble. Since, it is possible to transmit ultrasound pulses and immediately afterwards detect reflection signals from the bubbles within the ultrasound beam path with the same transducer. Measuring probes, looking like a simple stainless steel rod, can be constructed.
After a short ultrasound pulse with a frequency of 4 [MHz], for example, and a pulse length of about ten cycles was transmitted, the probe is switched to the detection mode. Then the reflections from the bubbles present in the beam path are recorded as a function of time. Those that arrive within a defined time interval can be assigned to a spatial segment of the beam profile, the measurement volume. Its size can be calculated from the sound propagation velocity in the liquid phase. The distance of the measuring volume from the probe can thus be adjusted electronically. Usually a distance of about 3 [cm] is taken in bioreactors. At this distance, the diameter of the measurement volume is about 1.5 [cm] and thus larger than the usually appearing bubbles. With such a technique we measure long-time-averages of the bubble velocities. When three sensors are arranged in such a way that they probe a common measurement volume, the mean velocity in each direction can be estimated from their signals. These mean bubble velocity patterns are cylindrically symmetric. Hence, to depict the pattern structure, it is sufficient to show only half of the pattern. Typical examples for the 200 [L] stirred tank reactor already taken as an accompanying example, are shown in figure 5. In this particular case, the reactor was equipped with three standard Rushton impellers with diameters D=T/3, i.e. of a third of the tank diameter T. An essential message from these detailed patterns is that the mean gas flow does not go straight through the vessel but depicts a zig-zag motion through the reactor. This is important to the gas residence time in the reactor and hence for the mass transfer efficiency. Further, the liquid flow cells around these radially transporting impellers are so strong that they are able to draw bubbles from above towards the impeller hub. Hence gas does not only enter the cavities behind the impeller blades from below. Importantly the induced liquid velocities around the impeller blades are smaller at the lower impellers, since their gas load is higher and hence their power input is lower.
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(i)
According to Clift et al. [33], the mean bubble rise velocity in aqueous solutions is roughly 20 [cm/s]. It is thus instructive to subtract from the velocities shown on part A in figure 5 this value from the vertical component of each vector. The result is shown on part B; it is a very good first approximation to the mean liquid velocity pattern in these reactors as was shown in Godo et al. [34]. This pattern clearly shows that flow recirculation cells develop around each impeller. Rushton turbines throw out the dispersion in radial direction and part of this mass is drawn back towards the hub of the impeller. This compartment formation has immediate consequences for the mixing of fluid added to the vessel. Another important consequence of the cavities formation is, that they do not only reduce the power input of the impeller operated at a given stirrer speed, they also may completely change the flow characteristic of the outflow from this pumping device. A typical example is shown in figure 5. Here, two different impeller systems are compared: In part (A) and (B) a standard arrangement with three Rushton turbines is shown. In part (C) three impellers are chosen where the upper two are down-pumping hydrofoils (Lightnin's A315). With this arrangement we initially wanted to introduce a more global circulatory flow through the entire reactor by means of the axially transporting impellers. In single phase reactors that worked. In a two-phase system, however, the flow characteristics of these impellers appeared to be completely different as shown in the figure. Instead of moving down, the bubbles between both down-pumping A315s were found to rise quickly. The reason for that effect is that the cavities formed behind the impeller blades, completely changed the outflow characteristics of the impellers. The straight downward flow observed in the single-phase case changed into a broad outflow characteristics in the air in water dispersion and we observe separate circulation cells around each of the hydrofoil impellers. This effect is surely not restricted to hydrofoil impellers as other impeller types such as pitched blades also suffer from cavity formation. The operational conditions in these examples are different to that usually applied in animal cell cultures. In these cases the effects are easier to show, but the same effects appear in the original operational domain. As already shown by Oh et al. [35], damage of animal cells merely by Rushton impellers is still overestimated in literature. It is now known that Rushton impellers can be used in practice and operated at quite high agitation rates [3,4]. Cell damage mainly takes place due to other effects. The mean velocity patterns show that the global fluid flows induced with various impeller systems in aerated bioreactors are quite complex. It should be kept in mind that these mean patterns do show only one aspect of the flow, the longtime average. The chaotic flow that is really seen by the cells is another important aspect as this mostly influences the mixing property of the reactor that is of key importance to local homogeneity. Mixing experiments are studied next.
3.2 Mixing of the continuous liquid phase
Most papers about bioreactors deal with liquid-phase mixing, since this problem is of critical importance [36]. In practice, additions of substrate components or corrective agents such as base or antifoam detergents are made locally. Most often the material is added at the top surface of the dispersion because of the fear about problems with aseptic operation and with cleaning [7]. This added material must quickly be distributed across the entire culture to reach homogeneity. This process is mainly dominated by the flow patterns in the reactor. Most intuitive information about the global mixing can be obtained from mixing time measurements, where the mixing time is the time required to achieve a predefined level of homogeneity in the bulk liquid of the entire reactor upon addition of a tracer pulse at a particular point in the vessel. Experimentally, the homogenization of locally added material can be analyzed in mixing time measurements. The inverse of the mixing time primarily characterizes the efficiency of the mixing process. Two different types of experiments can be performed to achieve the mixing time : global decolorisation experiments in transparent vessels local tracer measurements with probes in production vessels
(ii)
These two different methods will be described and discussed in the next sections.
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3.2.1 Decolorization experiments in experimental vessels
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In transparent reactor mockups, dye decolorization is a very instructive mixing time measurement technique. Due to the transparent walls the mixing time can be obtained globally by visual inspection of decolorisation reactions after one reactant is injected into the dispersion at one place. The decolorisation process can be recorded with a video camera and the mixing time can simply be measured with a stop clock. Most often the iodine-iodide change in the presence of starch is used. I2 + 2 Na2S2O3 2 NaI + Na2S4O6
The experimental technique using the iodide system is well established and used since many years. For such experiments, the entire liquid phase is first colored with iodine, and then thiosulfate is added which removes the dark color. Besides the mixing time itself this method also delivers information of local mixing behavior, i.e. poorly mixed regions can be identified as places where the colored component does not quickly react away.
At the upper part of figure 6 shows such an iod-iodine decolorisation experiment. The new engineering aspect is that we are now able to compute this single phase mixing process. An optimal testimonial that we understand the essential properties of the mixing process is that we can show that our computational results based on computational fluid dynamics match with the experimental results as shown in figure 6. The numerical as well as the real experiment was performed in a pilot reactor of 400 [L] working volume equipped with three Rushton impellers but also with three pitched blade impellers or combination of both types on the single shaft. The results shown in the figure 6 clearly indicate that the current programs considering turbulence effects are able to match the experimental results fairly well. The mixing time value extracted from these experiments is taken as the time at which 95% of the initial color can be recorded on the photo. The point we would like to make here is that CFD computations are now ready to describe important experiments needed to characterize the transport properties of bioreactors under unaerated conditions to a useful degree of accuracy. This allows to significantly reducing the number experiments necessary during bioreactor development. However, it does not at all make experiments obsolete, as every model must finally be validated with concrete accurate data before pricy consequences can be drawn from its solutions. Quantitatively, mixing was previously described by means of simple engineering correlations. Ruszkowski [37] and many others presented the mixing time (m) correlation
m = ( Tg D / T ) 3 T 3
1
(4)
where A is a factor (5.9 in Ruszkowskis work), Tg is the integral energy dissipation rate, i.e. the power introduced by agitation and aeration to the entire tank per mass of culture. T is the tank diameter, H the height of the dispersion and D the impeller diameter. Originally it was developed for aerated stirred tanks with an aspect ratio H/T=1 which seldom appears in large-scale production bioreactors. For larger aspect ratios H/T>1, it is claimed that the mixing time is proportional (H/D)2.43. The correlation is also restricted to single impellers. It essentially says that the mixing time only depends on the geometry and not on the agitation system for the entire energy dissipation rate interval 0.02 to 5 [W/kg] covering not only the animal cell culture applications but also other bioreactor tasks [38]. Eq. (4) is under debate (e.g. Marks [7]) as it often leads to misunderstandings since the correct application domain was not clear even to reviewers of the field. For the important applications in currently used bioreactors there is no alternative that considers the influence of the number of impellers and their transport characteristics. Tests with different agitation systems show that the different impeller systems currently used in production reactors lead to different mixing times under operating conditions which are close to those used in commercial production units. The original data in figure 7, obtained with Rushton turbines and pitched blade impellers, show that at the same power input the spatial decolorisation patterns and the speed of decolorisation are much different for both agitators settings. These results thus provide experimental evidence to the side remark made in Roels [39], namely that impellers inducing axial flow components reduce the mixing time by about a factor two and that this is not restricted to extremes such as high solidity ratio impellers such as the so-called elephant ears. The reason for this
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improvement is the notably improved top-to-bottom mixing by the more global flow structure induced by the axially down-pumping pitched blade impellers. The result from figure 7 is essentially that the different impeller configurations appear in the factor A of Eq. (4).
A closer visual inspection of the decolorisation results for three Rushton turbines, as depicted in figure 7, shows that the reaction appears to proceed in steps. In the case of top feeding of the thiosulfate, first of all the region about and above the upper impeller gets decolorized. Next the middle section and finally the area around the lower impeller become decolorized. This stepwise process points to compartment formation in the liquid flow. This fact that was already found by several authors e.g. Manikowski et al. [32], can also be explained with CFD results shown in figure 6.
The compartment formation and the decolorisation speed are much different when different impeller systems are taken. Figure 7 shows a quantitative result in form of scenes from a video and the quantitative mixing time measurements. The standard impeller set with Rushton turbines is compared with a set of pitched blade impellers, i.e. radial transporters with axial transporters. Pitched blade impellers can be considered a compromise between Rushton turbines and propellers or the hydrofoil impellers already mentioned, as they depict considerable radial flow components and show up power numbers that are roughly between that of marine propellers and Rushton turbines. For the arrangement shown in figure 7, the turbulent Power numbers we determined to Po = 15.3 for the set of Rushton turbines while the set of pitched blade impellers gave Po = 4.1 showing that a larger part of the energy supplied is channeled into directed fluid motion. When we follow up, in a numerical experiment, the path of an individual liquid particle in the flow in some Lagrangean view, then we see that the probability to escape the recirculation flow compartments around a single impeller into which they were placed is rather low. As can be seen from the results shown in figure 7, there is no direct circulation path. The individual cell in such an environment performs a rather chaotic motion in the turbulent flow field, which only upon long-time averaging (with time constants in the order of hours) gets the appearance of the regular circulation motion suggested in many textbooks. A similar result was already observed many years ago in bubble column reactors by a comparison between radioactively marked flow follower data and Lagrangean CFD simulations [40]. This message is important to scale down approaches which sometimes assume periodic movements of cells between zones with different environmental properties. It should be kept in mind that mixing times are no absolute quantities. They critically depend on the experimental conditions, particularly the point where the tracer or the reactant is added to the fluid flow. The results depicted in figure 8 show that the effect is quite large. The essential message from these results and similar data from Cronin et al. [41] for two Rushton turbines is that an addition between the impellers leads to the shortest mixing times. The consequence that should be drawn is to add substrates and correctives such as base for pH regulation to the culture in a distributed form or at least between the upper two impellers. So they will be distributed much faster across the entire culture or actually at several points at the same time. In industrial biotechnology, this fact is known for long time. Multiple substrate inputs were already implemented in very large reactors, e.g. ICIs tower loop reactor, but currently not in animal cell cultures as the responsible people fear clogging and sterility problems. Practitioners who dislike this advice should remember that one component needed to metabolize the substrate, namely the oxygen in air, is fed below surface since ever without severe practical problems.
3.2.2 Stimulus-response experiments in production-scale reactors

The decolorisation technique provides very good impressions on mixing of a reactant added to the reactor within the culture. Unfortunately this technique cannot be applied at production vessels due to regulatory cGMP constraints. The alternative is using tracer materials that commonly are in contact with the vessels. E.g. saline solutions or the solutions applied during the CIP procedures. We took the salt pulse technique and used conductivity probes as sensors. The results in terms of mixing times could be validated against the decolorisation measurement data. The positions of sensors were derived from the decolorisation experiments as the places where the decolorisation most sensitively changed upon changes in the operational conditions. The three sensors were placed near the wall of the vessel half way between the baffles. They were attached to the gas tube which supplies the sparger at heights of 0.2 [m], 2.7 [m], and 4.1 [m] above the reactor bottom with a clearance of 3 [cm] from the vessel wall.
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In previous investigations on the mixing times one usually added the tracer at the top and measured its concentration at the bottom as a function of time. The result is the so-called top-to-bottom mixing time. In order to get more detailed information about the mixing process it is straightforward to place further detectors into the reactor volume. One additional sensor was placed near the top surface of the dispersion opposite to the addition point to prevent wake effects. A further one was positioned half way between both. A typical result of such three-probe arrangement in a system of down pumping axial transporters can be seen in figure 9. From the response curves we can see again that the progress proceeds in steps which are due to the compartment effects discussed in the context of the decolorisation investigations.
A wealth of fluid dynamic information can be extracted from such response curves that at the first glance look as noisy curves. The down-pumping impellers pump the tracer added at the top close to the shaft immediately downwards so that the lowest probe responded first. The fastest top-to-bottom transport is roughly one tenth of the entire mixing time. The modulation frequency on the response curves from the middle and upper probe reflect compartment formations. Finally all curved end up at the same conductivity.
3.3 Oxygen mass transfer
The power transferred into the bioreactors via the agitator is also the key influence variable on the mass transfer from the dispersed gas-phase, into the continuous liquid phase of the culture. Several oxygen mass transfer measurement techniques have been developed in the past. The one that can easily be applied even to production bioreactors is the gassing-in/ gassing-out method, where the gas supply is switched for some time from pressurized air or pure oxygen to a gas that does not contain oxygen, e.g., pure nitrogen. Upon this change the dissolved oxygen concentration in the culture, measured with a standard dissolved oxygen probe drops. When the oxygen is completely stripped, the gas supply is switched back again to air and the corresponding rise in the dissolved oxygen concentration is recorded. A simple oxygen balance equation suffices to model this experiment. The key model parameter to be fitted to the data is the kLa value, the volumetric mass transfer coefficient. From the bioreactor performance point of view this is the key reactor characteristic for mass transfer.
With improved measurement techniques, particularly by replacing conventional Clark electrodes by modern fluorescence probes and automation of the measurement procedure with modern control techniques (e.g., Sieblist et al. [42]), the error in such measurements can be drastically reduced to less than 5% as compared to techniques employed in early experiments on the subject (e.g. Cooke et al. [43]) where the error was in the 20% range.
Figure 10 shows a new assessment to the dependency of the volumetric oxygen mass transfer coefficient kLa as a function of the specific mechanical power input to a 400 [L] reactor as measured with a torque meter on the impeller shaft. Measurements were performed in an aqueous electrolyte solution the osmotic pressure adjusted to the value of 300 [mOsmol], a typical value prevailing in animal cell cultures. The experiments were performed at different superficial gas velocities wsg and for two different impeller configurations, a single impeller and a set of three impellers. First it should be noticed in this double logarithmic representation that kLa exponentially depends on the specific power input, i.e. the plot is practically linear. Secondly we see that a higher gas throughput wsg leads to a higher kLa value. Usually it is assumed in literature [44, 45] that kLa essentially correlates with the integral power dissipation density and the integral aeration rate per reactor cross section, i.e., the superficial gas velocity wsg:
k L a = (P/V ) wsg
(5)
This equation is most often called as vant Riet-correlation and fits quit well for power input ranges used in cell culture bioreactors, as can be seen in figure 10.
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However, as should be thirdly noted in the results displayed in figure 10, the single impeller version leads to a significantly higher kLa value, at same superficial gas velocity in coalescence repressing medial (such as normal culture broths) as compared with the three-impeller configuration. This essentially says that in order to achieve a high mass transfer coefficient, the power input must be concentrated to the area where the bubble sizes and thus the specific interfacial area, a, is being formed. In other words the local power dissipation density max is of importance to mass transfer and not only the integral power transferred to the reactor liquid. Hence, we must be reminded to the fact, which was already shown by Oosterhuis & Kossen in 1985 [46], that even the oxygen is more or less locally transferred to the reactor and this needs intensive homogenization procedures as the low amounts of oxygen that can be solved in the liquid are consumed by the cells rather quickly, i.e., faster than it can be distributed across the entire culture. Oosterhuis and Kossen [46] moved a dissolved oxygen sensor along a stainless steel cable in a production reactor and found that the dissolved oxygen concentrations showed pronounced maxima at the impeller positions.
3.4 Heat removal

The oxygen consumption rate of the cells immediately leads to metabolic heat production and in order to keep the cultures at their temperature optimum the produced heat as well as the heat generated by the agitator or stirrer must be removed. A detailed discussion is provided by Palomares and Ramirez [5].
As pointed out in detail by Roels [39], practical estimates are sufficiently accurately described when one assumes a constant metabolic heat generation on oxygen consumption yield of Yho = 460 [kJ/mol(O2)] which, surprisingly, holds generally true for nearly all cellular species. With the oxygen consumption of even fast growing high cell density cultures, the heat generated can easily be removed in stirred tank reactors of up to volumes of 20 [m]. Opposite to microbial systems where internal heat exchanger tubes become necessary in high cell density cultures, bioreactors for cell cultures are able to remove the heat via cooling jackets around the cylindrical part of the vessels. Hence, heat removal does not count to the critical problems in bioreactor design and scale-up for cell cultures.
4 Concluding remarks
This paper shows some important aspects of large scale bioreactors that are now fairly well understood and can be modeled by means of computational fluid dynamics. Key variables can be measured and compared with the fluid dynamic model solutions in order to verify these models. Particularly the large-scale motion of fluids is quite well understood and can be simulated.
Mixing in the sense of obtaining sufficiently homogeneous conditions remains to be the most important task for the agitation system in animal cell culture reactors. The levels of power input per mass unit, P/W, that are applied with agitators in industrial cell culture fermenters in order to achieve reasonable mixing times are far below the levels where cell damage occurs. With the larger diameter impellers chosen, the tip velocities or shear stresses are about two orders of magnitude larger than the mean P/W but significantly lower than those that were found to be influencing the product quality. Reliable scale-up designs can only be expected from solutions of full three-dimensional nonlinear models of the twophase gas-liquid flow in bioreactors. Also for bioreactors the goal must be basing scale-up studies on detailed knowledge of the fluid dynamic details. With stirred tank reactors, we are currently often restricted to single-phase computations but we are proceeding in the way to full two-phase computations, which are already possible for bubble columns (Godo et al. [34]), with a couple of good software developments. Although much has been done so far, there is a long way ahead of us to solve that problem. It is not only the computational fluid dynamic that must be advanced, the same importance have further developments of experimental techniques that can supply the computations with detailed validation data. Without such a model validation we cannot dare to draw cost intensive consequences from CFD results. Moreover, we would be forced to make scale-up studies on rather shaky grounds.
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Finally we would like to state that we see a broad consensus with the extended work performed by Professor Nienow and his collaborators with respect to the effects essential to animal cell bioreactor fluid dynamics and their implications to bioreactor design and scale up. We would like to encourage young biochemical engineers in academia to continue this detailed work which currently seems being eclipsed by cell improvement aspects.
The authors have declared no conflict of interest
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P/V A a D dO2 H kL kLa L N P Po Q T V W wsg Yho max mean 95% , ,
Symbols used
[W/m] [-] [m/m] [m] [mg/L] [m] [cm/s] [s-1] [m] [rpm] = [1/min] [W] [-] [L/min] [m] [L or m] [kg] [m/h] [kJ/mol(O2)] [W/kg] [W/kg] [W/kg] [s] [s] [m] [kg/m] [m/s] [-] [W/kg] power input per volume engineering constant mixing time correlation specific gas-liquid interfacial area stirrer diameter dissolved oxygen concentration height of the dispersion oxygen transport coefficient volumetric oxygen transfer coefficient edge length in CFD model stirrer rotation speed power Power Number gas flow rate tank diameter volume weight superficial gas velocity oxygen consumption yield energy dissipation per unit mass maximum energy dissipation per unit mass mean energy dissipation per unit mass mixing time mixing time for 95% homogeneity smallest eddies size density per unit mass kinematic viscosity constants for vant Riets correlation mean energy dissipation per unit mass
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[1] [2] [3] [4] [5] [6] [7] [8] [9]
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Nienow, A.W., Reactor engineering in large scale animal cell culture, Cytotechnology, 2006, 50, 9-33 Nienow, A. W., Impeller Selection for Animal Cell Culture. in: Flickinger, M. C. (Ed.), Encyclopedia of Industrial Biotechnology, Wiley, New York, 2010 Palomares, L.A., Ramirez, Bioreactor scale-up, in: Flickinger, M. C. (Ed.), Encyclopedia of Industrial Biotechnology, Wiley, New York, 2010
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Marks, D., Equipment design considerations for large scale cell culture, Cytotechnology, 2003, 42, 21-33 Bylund, F., Collet, E., Enfors, S.O., Larsson, G., Substrate gradient formation in the large-scale bioreactor lowers cell yield and increases byproduct formation, Bioproc. Eng., 1998, 18, 171-180 Serrato, J.A., Palomares, L.A., Meneses-Acosta, A., Ramirez, O.T., Heterogeneous Conditions in Dissolved Oxygen Affect N-Glycosylation but Not Productivity of a Monoclonal Antibody in Hybridoma Cultures, Biotechnol. Bioeng., 2006, 88, 176-188
[10] Murhammer, D. W. & Goochee, C. F., Sparged Animal Cell Bioreactors: Mechanism of Cell Damage and Pluronic F-68 Protection, Biotechnol. Prog., 1990, 6, 391-397 [11] Jordan, M., Eppenberger, H. M., Sucker, H., Widmer, F. Einsele, A., Interactions between animal cells and gas bubbles: The influence of serum and pluronic F68 on the physical properties of the bubble surface, Biotechnol. Bioeng., 1994, 43, 446-454 [12] Chattopadhyay, D., Rathman, J. F., Chalmers, J. J., The protective effect of specific medium additives with respect to bubble rupture, Biotechnol. Bioeng., 1995, 45, 473-480
[13] Al-Rubeai, M, Sing, R. P., Goldman, M. H., Emery, A. N., Death mechanisms of animal cells in conditions of intensive agitation, Biotechnol. Bioeng., 1995, 45, 463-472
[14] Gray, D.R., Chen, S., Howarth, W., Inlow, D., Maiorella, B.L., CO2 in large-scale and high-density CHO cell perfusion culture, Cytotechnology, 1996, 22, 6578 [15] vant Riet, K., Tramper, J., Basic Bioreactor Design, Marcel Dekker, New York, 1991 [16] FDA (2011), Process Validation: General Principles and Practices, FDA Guide for Industry, Revision 1, 20.1.2011, 1-19 [17] Sieblist, C., Hgeholz, O., Aehle, M., Jenzsch, M., Pohlscheidt, Lbbert, A., Insights into Large Scale Cell Culture Reactors: II. Gas-Phase Mixing and CO2 Stripping, Biotechnology Journal, in press DOI: 10.1002/biot.201100153
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[18] Henzler, H. J., Homogenisieren - Referenzsysteme und Methoden zur Erfassung der Homogenisierungseigenschaften von Rhrsystemen, GVC-Fachausschu Mischvorgnge, 1998
[19] Garcia-Ochoa, F. & Gomez, E., Oxygen Transfer Rate Determination: Chemical, Physical and Biological Methods, in: Flickinger, M. C. (Ed.) Encyclopedia of Industrial Biotechnology, Wiley, New York, 2010 [20] Kolmogorov, A. N., The local structure of turbulence in incompressible viscous fluid for very large Reynolds numbers., Proceedings of the USSR Academy of Sciences, 1941, 30, 299303. [21] Bakker, A., Oshinowo, L.M., Modelling of turbulence in stirred vessels using large eddy simulation., Chem. Eng. Res. Des., 2004, 82, 1169-1178
[22] Van den Akker, H.E.A., The details of turbulent mixing process and their simulation, Adv. Biochem. Eng. Biotechnol., 2006, 31, 151-229
[23] Behrendt, U., Szperalski, B., Engineering aspects for the production of recombinant proteins from animal cell cultures, Presentation 11th European Congress of Biotechnology, Basel, 2003 [24] Alcamo, R., Micale, G., Grisafi, F., Brucato, A., Michele Ciofalo, M., Large-eddy simulation of turbulent flow in an unbaffled stirred tank driven by a Rushton turbine, Chem. Eng. Sci., 2005, 60, 2303 2316
[25] Bche, W., Leistungsbedarf von Rhrwerken, Z.VdI, 1937, 81, 1065 [26] Myers, K., Reeder, M.E., Fasano, J.B. (2002), Optimize mixing by using the proper baffles, Chem. Eng. Progr., 2002, 98, 42-47
[27] Lu, W.-M.; Wu, H.-Z. & Ju, M.-Y., Effects of baffle design on the liquid mixing in an aerated stirred tank with standard Rushton turbine impellers, Chem. Eng. Sci., 1997, 52, 3843-3851
[28] Meier, S., Cell culture scale-up: Mixing, mass transfer and use of appropriate scale-down models, at: Biochemical Engineering XIV, Harrison Hot Springs, B.C., Canada, July 2005
[29] van't K. Riet, Turbine agitator hydrodynamics and dispersion performance, Dissertation, Delft University of Technology, 1975 [30] van't Riet, K. and J. M. Smith, The trailing vortex system produced by Rushton turbine agitator, Chem. Eng. Sci., 1975, 30, 1093
[31] Brring, S., Fischer, J., Korte, T., Sollinger, S. and Lbbert, A., Flow structure of the dispersed gasphase in real multiphase chemical reactors, investigated by a new ultrasound Doppler technique. Can. J. Chem. Eng., 1991, 69, 1227-1257 [32] Manikowski, M., Bodemeier, S., Lbbert, A., Bujalski, W., Nienow, A. W., Bubble velocity patterns in aerated stirred tank reactors, Can. J. Chem. Eng., 1994, 72, 769-781 [33] Clift, R., Grace, J.R., Weber, M.E., Bubbles, drops, and particles, Academic Press, San Diego, 1978
[34] Godo, S., Junghans, K., Lapin, A., Lbbert, A., Dynamics of the flow in bubble columns, in: Sommerfeld, M. (Ed.), Bubbly Flows, Analysis, Modelling and Calculation, Springer, Berlin, 2004, 53-66 [35] Oh, S.K.W., Nienow, A.W., Al-Rubeai, M., Emery, A.N. , Further studies of the culture of mouse hybridomas in an agitated bioreactor with and without continuous sparging, J. Biotechnol., 1992, 22, 245 270 [36] Langheinrich, C., Nienow, A.W., Control of pH in Large-Scale, Free suspension animal cell bioreactors: alkali addition and pH excursions, Biotechnol. Bioeng., 1999, 66, 171-179
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[37] Ruszkowski, S., A rational method for measuring blending performance and comparison of different impeller types., in Proc. 8th European Mixing Conference, Institution of Chemical Engineers, Rugby U.K, 1994, 283-291. [38] Nienow, A. W., On impeller circulation and mixing effectiveness in the turbulent flow regime, Chem. Eng. Sci., 1997, 52, 2557-2565
[39] Roels, J.A., Energetics and kinetics in biotechnology, Elsevier Biomedical Press, Amsterdam, 1983 [40] Devanathan, N., Dudukovic, M., Lapin, A., Lbbert, A., Chaotic flow in bubble column reactors, Chem. Eng. Sci., 1995, 50, 2661-2667
[41] Cronin, D. G., Nienow, A.W. Moody, G.W., An experimental study of the mixing in a protofermenter agitated by dual Rushton turbines, Food and Bioprod. Proc., Trans. I. Chem. E., Part C, 1994, 72, 35-40 [42] Sieblist, C., Aehle, M., Pohlscheidt, M., Jenzsch, M., Lbbert, A., A test facility for fritted spargers of production-scale-bioreactors, Cytotechnology, 2011, 63, 49-55 [43] Cooke, M., Middleton, J.C., Bush, J.R., Mixing and mass transfer in filamentous fermentations, in: R. King (Ed.) Proc. 2nd International Conference of Bioreactor Fluid Dynamics, BHR Group, Cranfield, UK, 1988, 37-64 [44] Riet, K. v.;. Review of measuring methods and results in nonviscous gas-liquid mass transfer in stirred vessels, Ind. Eng. Chem. Proc. Des. Dev., 1979, 18 (3), 357-364 [45] Xing, Z.; Kenty, B. M.; Li, Z. J. & Lee, S. S., Scale-up analysis for a CHO cell culture process in largescale bioreactors, Biotechnol. Bioeng., 2009, 103, 733-746 [46] Oosterhuis, N.M.G., Kossen, N.W.F., Modelling and scaling-up of bioreactors. in: Brauer, H. (Ed.), Biotechnology 2, VCH, Weinheim, 1985
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6 Figure Legends
Fig. 1 Photos from fully baffled production reactors for animal cultures equipped with three Rushton turbines (A) and three pitched blade turbines (C) [22]. For comparison CFD simulations of the mean ungassed flow patterns developing in such reactors are shown (B, D)
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Fig. 2 (A) Formation of a cavity behind a blade of a Rushton turbine upon starting the aeration. The series of pictures are scenes from a video observed with a CCD camera rotating with the impeller. (B) CFD visualization, made by A. Bakker, ANSYS Inc., of the vortices developing at the upper and the lower edge of Rushton turbine blades which attract the gas flow around the blades leading to gas cavities. (published with permission)
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Fig. 3 Cavities forming in the vortices behind so-called elephant ear impellers at a power input of 0.03 [W/kg] in a 400 [L] experimental vessel (T= 640 [mm]; D/T=0.33). Here the main cavity develops at the upper edge of the impeller only. (A: beginning of the development at low gas throughputs (0.009 [vvm]); B: more voluminous cavity at 0.019 [vvm]; C: large cavitiy formation on top of the impeller blade with 0.028 [vvm] gas throughput.)
Fig. 4 Power Number drawn by a set of Rushton turbines in a 10 [m] production reactor for cell culture as a function of the stirrer speed and the aeration rate. The draw is shown relatively to the unaerated case. For a given impeller speed the Po-number drops with increasing aeration rate and vice versa.
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Fig. 5 Liquid and bubble velocity patterns in a 200 [L] stirred tank reactor aerated with a ring sparger at the bottom of the vessel (o). The positions where the velocities were measured are indicated by the dots with lines representing the velocity vectors. Only one half of the reactor is shown. (A, B) The agitator shaft is equipped with three Rushton turbines. (A) Estimate of the liquid phase velocity pattern obtained by subtracting a vertical velocity component of 20 [cm/s] from the measured bubble velocity vectors (B). The impeller speed was N=400 [rpm] and the aeration rate Q=0.33 [vvm]. (C) Bubble velocity pattern for a configuration of agitator shaft equipped with two downward pumping hydrofoil impellers and at the bottom above the sparger a single Rushton turbine [30]. The impeller speed was N=300 [rpm] and the aeration rate Q=0.36 [vvm].
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Fig. 6 Comparison of CFD simulation (lower row) and experimental results (upper row) from iodide decolorisation method in a 400 [L] pilot scale model reactor equipped with three standard Rushton turbines operated at P/V = 90 [W/m]. The CFD simulation only shows a cut through the reactor containing the impeller shaft.
Fig. 7 Mixing of 400 [L] vessel equipped with three Rushton turbines in comparison to three pitched blade impellers. Left: decolorization characteristics upon addition of thiosulfate to the top surface near the shaft Right: mixing time results with fitting of Eq. (4) (dashed line) (A3xRT=17.9; A3xPB=10.5).
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Fig. 8 Mixing times measured in a 400 [L] stirred tank reactor operated at 90 [W/m] where the tracer was added to the system at various points. The numbers on the bars show the difference in mixing time compared to the reference addition at the top of the liquid surface.
Fig. 9 Stimulus response curves from 12 [m] unaerated stirred tank cell culture reactor operated at 50 [W/m] mechanical power input. Tracer addition at the top surface, responses measured with three conductivity probes at the top, in the mid and at the bottom of the tank closed to the reactor wall.
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Fig. 10 (A) kLa as a function of the superficial gas velocity wsg and the specific power input P/V induced by the impeller system. The symbols represent the measurement results. The hypersurface of vant Riet-correlation [44] is fitted to experimental data. Lower row: Comparison of kLa values for a 400 [L] cell culture bioreactor equipped with two different stirrer types (Rushton turbines (B); pitched blade impellers (C)) as a function of power input and superficial gas velocity. Experiments were performed using a single stirrer compared to a 3-stage impeller configuration.

Liquid Mixing and Oxygen Supply

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www.biotechnology-journal.

cell culture, oxygen mass transfer, mixing, large scale bioreactors

2 Materials and methods

Ptotal = Pstirrer + Pgas + Pfriction

3 Results and discussion

3.2 Mixing of the continuous liquid phase

3.2.1 Decolorization experiments in experimental vessels

3.2.2 Stimulus-response experiments in production-scale reactors

3.3 Oxygen mass transfer

3.4 Heat removal

The authors have declared no conflict of interest

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