Shake-Flask Bioreactors

2.17

Shake-Flask Bioreactors

W Klöckner and J Büchs, RWTH Aachen University, Aachen, Germany © 2011 Elsevier B.V. All rights reserved.

2.17.1 2.17.2 2.17.3 2.17.3.1 2.17.3.2 2.17.4 2.17.4.1 2.17.4.2 2.17.5 2.17.5.1 2.17.5.2 2.17.5.3 2.17.5.4 2.17.6 2.17.6.1 2.17.6.2 2.17.6.3 2.17.6.4 2.17.7 2.17.7.1 2.17.7.2 2.17.7.3 2.17.8 2.17.9 2.17.9.1 2.17.9.2 2.17.9.3 References

Introduction Specific Power Input in Shake Flasks Out-of-Phase Phenomena in Shake Flasks Relevance of the Out-of-Phase Phenomena for Screening Calculation of Out-of-Phase Conditions Maximum Energy Dissipation Rate in Shake Flasks Relevance of Hydromechanical Stress in Shake Flasks Calculation of the Maximum Local Energy Dissipation Rate in Shake Flasks Gas/Liquid Mass Transfer in Shake Flasks Experimental Setup for Determination of Maximum Oxygen Transfer Capacity Influence of Flask Wall Material on Liquid Film and Oxygen Transfer Influence of Operation Conditions on Oxygen Transfer Mass Transfer Resistance of Sterile Plugs Baffled Shake Flasks Specific Power Input in Baffled Shake Flasks Out-of-Phase Phenomena in Baffled Shake Flasks Maximum Energy Dissipation Rate in Baffled Shake Flasks Oxygen Transfer in Baffled Shake Flasks Use of Engineering Parameters for Scale-Up from Shake Flask to Stirred-Tank Reactor Specific Power Input as Scale-Up Parameter Maximum Energy Dissipation Rate as Scale-Up Parameter Scale-Up of Ventilation from Shake Flask to Stirred-Tank Reactor Fed-Batch and Continuous Cultures in Shake Flasks Online Measuring Techniques in Shake Flasks Online Measuring Techniques of the OTR and Carbon Dioxide Transfer Rate Online Measurement of the DOT Online Measurement of the pH Value

Glossary computational fluid design (CFD) A computation program for the modeling and calculation of flow conditions. hydromechanical stress Parameter for the stress that acts on microorganisms due to fluid motion and pressure fluctuations. kLa Mass transfer coefficient for the gas/liquid mass transfer. maximum energy dissipation Maximum local energy input in the liquid flow.

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oxygen transfer rate (OTR) Amount of oxygen transferred per time and fluid volume through the gas–liquid interface. power input Describes the energy introduced into a bioreactor, which generates liquid flow and mixing and is finally dissipated as heat. scale-up Transfer of process conditions to a larger scale. Experiments are often conducted on a small scale to save costs. The optimized process conditions are afterward transferred to production scale. shake flasks A conical laboratory flask for the cultivation of microorganisms in a shaker incubator.

2.17.1 Introduction Shaken bioreactors are used as fermentation systems since the beginning of the last century. Probably the first submersed fermenta­ tion in a shake flask was realized by Kluyver and Perquin in 1933 [13]. Nowadays, shaken bioreactors are used for a considerable variety of tasks. Most common is the use of conventional conical Erlemeyer shake flasks with a nominal flask volume from 25 ml up to 6 l. These flasks are mainly used for screening and early bioprocess development in industry and academic research. One reason for the popularity of shake flasks as bioreactors is the simplicity of the system that enables the performance of a high number of costefficient parallel fermentations on a small scale. In spite of its wide use, the importance of engineering characteristics such as oxygen

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Bioreactors – Design

transfer and volumetric power input in shake flasks had been underestimated for a long time. This becomes obvious by comparing the extensive description of stirred-tank reactors in research papers with the few reports dealing with engineering characteristics of shake flasks. However, a detailed understanding of a fermentation process in a stirred-tank reactor will be without any benefit, if a previous screening has been conducted under obscure cultivation conditions. Furthermore, it will lead to high costs to reverse the negative outcome on production scale, if the screening experiments were performed under unsuited conditions. Basic engineering characteristics of shake flasks are discussed in the following pages. Thereby, this article aims to support engineers and biologists with fundamental knowledge about physical properties in shaken reactor systems and to provide suggestions on how to optimally apply shake flasks.

2.17.2 Specific Power Input in Shake Flasks The specific power input per liquid volume (P/V) is one of the essential parameters to specify cultivation conditions of micro­ organisms in shake flasks or stirred-tank bioreactors. Many physical reactor characteristics as, for example, hydromechanical stress or heat transfer, are directly related to the volumetric power input. Therefore, it is regarded as one of the crucial values for optimization and scale-up of culture conditions [59]. The quantity ‘specific power input’ describes the overall power introduced into a bioreactor, which generates liquid flow and mixing and is finally dissipated as heat as a result of friction in the fluid flow of the reactor. Two different torque methods and a heat balance method were applied for the measurements of the specific power input [11, 48, 49]. A comparison of the torque method and the heat balance method showed a satisfactory agreement of both methods, proving their reliability [49]. Figure 1 shows a graph of the specific power input on shaking frequency for different viscosities. As an example, a filling volume of 40 ml is chosen. In general, power input values in shake flasks are of the same order of magnitude as in stirred-tank reactors at typical operating conditions used for bacterial and yeast cultures (1–10 kW m−3). Specific power input per volume (P/V) in shake flasks is increasing with increasing shaking frequency and decreasing with increasing relative filling volume. The shaking diameter has no significant effect on the power input per volume (P/V) as long as the system is operating ‘in phase’ [8, 11]. The specific power input increases with viscosity. However, in the example shown in Figure 1, specific power input does no longer increase at viscosities larger than about 60 mPa s. At higher viscosities, even specific power input decreases. This is the result of unfavorable ‘out-of-phase’ operation conditions, which is discussed in Section 2.17.3. To get a concise understanding of hydrodynamic phenomena in bioreactors, it is advised to develop a dimensionless representa­ tion of the results [8]. The mechanical power of a rotating stirrer can be described as the product of torque (M) and angular velocity (ω). In addition, torque can be considered as a product of force (F) and lever arm (r): P ¼ M ⋅ω ¼ F ⋅r ⋅ω

½1

Power input in shake flasks occurs because of the friction between the liquid and the glass wall. The friction force (F) at the glass wall can be obtained by multiplying the wall shear stress (τW) with the corresponding friction area (AW). Consequently, P ¼ F⋅r ⋅ω ¼ τ W ⋅AW ⋅r ⋅ω

½2

Shear stress at the flask wall is a function of the fluid flow and can be calculated as follows [54]:

Specific power input (P/V) (kW m–1)

8 198.0 mPa s

7

104.2 mPa s 6

61.8 mPa s 32.3 mPa s

5

17.8 mPa s 0.8 mPa s

4 3 2 1 0 80

120

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360

400

Shaking frequency (n) (1/min) Figure 1 Specific power input on shaking frequency for different viscosities, 250-ml shake flasks, 40-ml filling volume, and 25-mm shaking diameter.

Shake-Flask Bioreactors τ W ¼ C1 ⋅ρ⋅r 2 ⋅ω2 ⋅ f ðReÞ

215 ½3

with the constant C1 and the liquid density ρ. The Reynolds number (Re) is defined as: Re ¼

ρ ⋅n ⋅d2 η

½4

with the dynamic viscosity of the liquid η. The maximum inner flask diameter (d) is a suitable characteristic length scale for the formation of the Reynolds number [8]. In contrast, Kato et al. [27] used the shaking diameter d0 as a characteristic length scale for the formation of the Reynolds number. The friction area AW in eqn 2 can be calculated with a model for the liquid distribution in shake flasks for liquids with low viscosities [12]. This model allows the exact calculation of the contact area between liquid and flask wall depending on shaking speed, flask geometry, shaking diameter, and filling volume. However, the detailed calculation is complex and the result cannot be expressed as an explicit equation. Due to the fact that the friction area (AW) is not significantly changing with shaking speed and shaking diameter (Büchs et al., 1995), the contact area can be described in simplified form as a product of flask perimeter (U = 2·π·r) and liquid height (h): AW ¼ 2 ⋅π⋅r ⋅h

½5 1=3 VL ),

depending on the total liquid volume (VL) [8]. The liquid height (h) can be described as a characteristic length scale (h ¼ From eqns 2, 3, and 5 and with the definition of the angular velocity (ω = 2·π·n) as well as the shake-flask diameter (d = 2·r), it follows that: 1=3

P ¼ C1 ⋅ π 4 ⋅ f ðReÞ ⋅ ρ⋅n3 ⋅ d4 ⋅VL

½6

By introducing a modified Newton number (Ne′): Ne′¼

P 1=3 ρ⋅n3 ⋅d4 ⋅VL

½7

eqn 6 can be transformed to: Ne′¼

P 1=3

ρ⋅ n3 ⋅ d4 ⋅VL

¼ C1 ⋅ π 4 ⋅ f ðReÞ

½8

In case of turbulent flow in unbaffled shake flasks, the correlation for turbulent flow conditions f(Re) = C2·Re−0.2 [54] can be introduced in eqn 8. Fluids with elevated viscosities lead to lower Reynolds numbers where transitional flow is prevailing. For this reason, a term for laminar flow (Re−1) and transitional flow (Re−0.6) is additionally added to eqn 8 [9]. This leads to the following equation: Ne0 ¼CLa ⋅Re− 1 þ CTr ⋅Re− 0:6 þ CTu ⋅Re− 0:2

½9

The resulting function includes three fitting parameters for laminar (CLa), transitional (CTr), and turbulent flow (CTu). Equation 10 is fitted to measuring points, covering from 25 ml to 5 l shake flasks with a nominal filling volume from 2% to 40%, by using a least square error method [46]. Only measuring points (more than 1000 in number) were considered at which the liquid bulk in the flasks is circulating ‘in-phase’ with the shaking table. A detailed description of the in-phase and out-of-phase phenomena is given in Section 2.17.3. From eqn 9 follows with the experimentally determined fitting parameters: Ne′¼70 ⋅Re− 1 þ 25 ⋅Re− 0:6 þ 1:5 ⋅Re− 0:2

½10

Figure 2 shows the correlation between the fitting function (eqn 10) and the experimentally determined data points [9]. An interesting conclusion of power input measurements in shake flasks is the fact that power input is increasing with increasing flask size for constant relative filling volume (VL/d3 = const.) and otherwise equal conditions. This occurs although the specific friction area (AW/VL), responsible for the power input, decreases. The reason for this is that the relative velocity between liquid and glass wall is increasing with increasing flask diameter. In contrast, the maximum oxygen transfer capacity is decreasing with increasing flask size for a constant relative filling volume [33]. Therefore, gas/liquid mass transfer is not directly correlated to specific power input in shake flasks, which is a significant difference to stirred-tank reactors. Power input in shake flasks was also analyzed by using computational fluid dynamics (CFDs) [Mehmood et al., 2009, 41, 69]. Zhang et al. [68] reported values for the power input determined by CFD calculation in the range of 0.04–0.6 kW m−3. These values are roughly one order of magnitude lower than experimentally determined values with different measuring techniques [8, 50, Peter et al., 2006] and therefore not reliable. In contrast to this, CFD simulations conducted by Ottow et al. [41] and by Mehmood et al. show a satisfactory agreement with experimental results. Applications of the power input correlation according to eqn 10 for hydromechanical stress analysis and for scale-up are described in Sections 2.17.4 and 2.17.7.

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Bioreactors – Design

101

Ne′ = 70 Re–1 + 25 Re–0,6 + 1.5 Re–0,2

‘In-phase’ operating conditions

Ne′ =

P

1010

ρ n3 d 4 VL1/3

(–)

100-ml shaking flask 250-ml

"

300-ml

"

500-ml

"

1000-ml

"

2000-ml

"

‘out-of-phase’ operating conditions

10–1

10–2 101

102

103

Re =

n⋅d 2⋅ρ

η

104

105

106

(–)

Figure 2 Modified Newton number (Ne′) on the flask Reynolds number (Re) for different flask size, viscosity (0.8–200 mPa s), shaking diameter (2.5–5 cm), and shaking frequency (80–400 rpm). The ‘in-phase’ conditions are labeled with large symbols and ‘out-of-phase’ conditions with small cross symbols.

2.17.3 Out-of-Phase Phenomena in Shake Flasks 2.17.3.1

Relevance of the Out-of-Phase Phenomena for Screening

Screening for improved culture conditions plays a decisive role in bioindustry. Selection of specific strains, medium development, and optimization of growth and product formation are only a few examples that require experimental investigations, which are usually done in shake flasks. Defined and reproducible experimental conditions are absolutely necessary requirements for successful screening experiments. Especially, a sufficient oxygen supply is crucial for growth and product formation of most industrially used aerobic cultures. During the cultivation of organisms with a fermentation broth of low viscosity, the liquid rotates in phase with the movement of the shaker [11, 45]. An elevated viscosity of the fermentation broth, as it usually occurs during the cultivation of filamentous organisms or in biopolymer fermentation, can lead to an out-of-phase condition [11]. This operation condition is characterized by a strong reduction of the rotational fluid movement. The main part of the liquid remains at the bottom of the flask, instead of rotating in phase with the movement of the shaker drive. Out-of-phase operation leads to a decreased power input and a reduced mixing and oxygen transfer [9, 11, 30, 45]. Consequently, the out-of-phase phenomena might lead to oxygen-limited cultivation conditions and, thus, will cause an undesired selection pressure toward a low-viscosity fermentation broth [45]. That means, in case of a filamentous microorganism, mutants with an altered morphology with less branching and shorter hyphae length tend to be selected. Consequently, operation conditions may change into in-phase operating conditions for these mutants, what leads to a better oxygen supply of the culture and, therefore, to a higher product formation compared to the reference strain, although the selected strain need not necessarily have an improved pathway for product formation. The same problem may occur in medium development. Under out-of-phase operating conditions such nutrient compositions of the media are favored, which result in low viscosity of the fermentation broth [45]. These media will not coincide with media that are optimal for production at suitable in-phase operation conditions.

2.17.3.2

Calculation of Out-of-Phase Conditions

To determine an out-of-phase operation in shake flasks, Büchs et al. [8] introduced a nondimensional phase number: Ph ¼

d0 ⋅½1 þ 3 ⋅log 10 ðRe f Þ > 1:26 d

½11

with the film Reynolds number,
Re f ¼

vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 u ! 32  1=3 2 ρ ⋅n ⋅d2 π4 u 4 V L 5 ⋅ 1−t1− η 2 π d

½12

Shake-Flask Bioreactors

217

The variables in eqns 11 and 12 are defined as follows: d0 is the shaking diameter, d the maximum inner flask diameter, ρ the liquid density, n the shaking frequency, η the dynamic viscosity, and VL the liquid volume. In addition, an axial Froude number was defined as precondition for the application of eqn 11 [8]: Fra ¼

ð2 ⋅π⋅nÞ2 ⋅ d0 > 0:4 2⋅ g

½13

with the shaking diameter d0, the shaking frequency n, and the gravitational acceleration g. The cultivation conditions can be considered as in phase for values of Fra > 0.4 and Ph > 1.26 according to eqns 11 and 13 [8]. Experiments that are carried out on normal shakers with shaking diameters of 25 or 50 mm with shake flasks of a nominal volume of less than 1 l can only reach out-of-phase operation for elevated viscosities of the fermentation broth [11]. Out-of-phase operation may, therefore, occur for fermentations with elevated liquid viscosity, caused by a filamentous growth of the micro­ organisms or the release of metabolic products such as biopolymers. For these cases, it is recommended to calculate the phase number using eqns 11 and 12 to ensure that the liquid is rotating in phase [30] and to avoid the mentioned disadvantages of an outof-phase operation. In case of high viscosity and a calculated phase number of Ph < 1.26 the most pragmatic way to increase the phase number is to use a shaker with an increased shaking diameter (d0 ≥ 5 cm). Also, an increase of shaking frequency and filling volume is partially effective to increase the phase number. Both measures lead to an increase of the centrifugal force acting on the bulk liquid and, thus, assist to overcome the viscous forces.

2.17.4 Maximum Energy Dissipation Rate in Shake Flasks 2.17.4.1

Relevance of Hydromechanical Stress in Shake Flasks

Hydromechanical stress can become a decisive parameter for growth and product formation of sensitive organisms as animal or plant cell cultures. It has been reported that the morphology of filamentous organisms grown in shake flasks differs from the morphology of organisms grown in stirred-tank reactors because of the obvious differences in hydromechanical stress in both fermentation systems [14, 21, 25]. Pellets are used to be significantly larger in shake flasks than in stirred-tank reactors at typical operating conditions. The reason for the fundamental difference in the level of hydromechanical stress was not known until recently. It was speculated in the past that the specific power input in shake flasks is lower than in stirred tanks. However, this has been shown to be wrong (see Section 2.17.2). Problems often occur during scale-up of a shear-sensitive bioprocess from shake flasks to stirred reactors [20]. Consequently, a successful scale-up requires the characterization of the hydromechanical stress in shaken bioreactors. The maximum local energy dissipation rate εmax can be used to quantify the intensity of hydromechanical stress in fermentation systems [20].

2.17.4.2

Calculation of the Maximum Local Energy Dissipation Rate in Shake Flasks

The average energy dissipation rate in a stirred-tank reactor can be described by: εf ¼

P Ne ⋅n3 ⋅ d5 ¼ V ⋅ V

½14

Liepe et al. [29] proposed a general relationship to quantify the maximum energy dissipation rate in a dispersing apparatus: εmax ¼ 0:1⋅

u3 h1

½15

with h1 as characteristic length and u as the velocity of the turbulence generating element relative to the liquid. In a stirred-tank reactor, the height of the stirrer blade is used as characteristic length (h1) and the tip speed (u = π·n·d) of the stirrer as velocity relative to the fluid flow. It has to be considered that fully developed isotropic turbulence is a necessary requirement for the application of eqn 15 [29]. For the characterization of the maximum energy dissipation rate in shake flasks, Peter et al. [47] roughly classified the liquid flow in unbaffled shake flasks into two regimes. According to the definition of the Reynolds number given in eqn 4, turbulent flow is prevailing in shake flasks above a critical Reynolds number of Recrit = 60 000 [47]. For Re > 60 000, the maximum energy dissipation rate εmax in unbaffled flasks can be calculated according to eqn 15 with the maximum liquid height h1 as characteristic length. For Re > 60 000, the maximum energy dissipation rate εmax can be considered as equivalent to the average energy dissipation rate (εmax = εf). The maximum liquid height h1 can be calculated with a model for the liquid distribution in shake flasks [12]. Although the model enables the accurate determination of the liquid height, the calculation is complex and very extensive. To simplify the determination of the maximum liquid height, Peter (2006) proposed two empirical correlations. Considering the value of the axial Froude number, defined as: Fra ¼

ð2⋅π⋅nÞ2 ⋅d0 2⋅g

½16

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Bioreactors – Design

with the shaking diameter d0, the shaking frequency n, and the gravitational acceleration g, the maximum liquid height can be calculated by: ⋅d − 0:11 ⋅n0:44 ⋅VL0:34 Fra > 0:4 h1 ¼ 1:11 ⋅d0:18 0 Fra < 0:4

h1 ¼

½17

1:31 ⋅d0:28 ⋅d0:02 ⋅n0:9 ⋅VL0:35 0 1 s ;

½18

with the following dimensions of the variables: h1 = [m]; d0 = [m]; d = [m]; n ¼ ½ VL = [m ]. Peter [46] reported a deviation of maximal 10% between the calculated values using eqns 17 and 18 and the calculated values using the liquid distribution model according to Büchs et al. [12]. Comparing the ratio of maximum to average energy dissipation rate (εmax/εf) between shake flasks (2–7) and stirred reactors (30–100), the ratios in shake flasks are more than one order of magnitude lower [47]. As the specific power input, which corresponds to the average energy dissipation rate εf, is more or less similar in shake flasks and stirred tanks, at typical operating conditions, the absolute values of the different ratio εmax determine the fundamental difference in hydromechanical stress of both reactor systems. This different ratio and, therefore, different distribution of power input in the liquid is the rational explanation for the empirical observation made since many decades that pellets in shake flasks tend to be significantly larger than in stirred tanks. The level of hydromechanical stress is the engineering parameter with the largest general difference between shake flasks and stirred tanks. Therefore, the scale-up of biological systems, which are sensitive with respect to hydromechanical stress, is a big challenge. 3

2.17.5 Gas/Liquid Mass Transfer in Shake Flasks A sufficient oxygen supply is crucial for a successful cultivation of microbial cultures in shake flasks. Growth and product formation become oxygen limited if the oxygen demand exceeds the maximum oxygen transfer capacity of the flask. In that case, the growth rate and product formation rate are only dependent on the oxygen transfer capacity and important growth parameters remain unrecognized. Also, shifts of metabolic pathways, caused by the oxygen limitation, have to be expected. Consequently, a sufficient oxygen supply is a precondition to investigate the effect of variables as, for example, media composition on growth and product formation [34]. To investigate the maximum oxygen transfer capacity of shake flasks, the mass transfer resistance capacity of the flask closure and of the gas/liquid interface has to be considered [37].

2.17.5.1

Experimental Setup for Determination of Maximum Oxygen Transfer Capacity

Different methods for the determination of the maximum oxygen transfer capacity are reported in literature. Veglio et al. [64] and Nikakhtari and Hill [38] reported on dynamic methods that require the measurement of the dissolved oxygen tension (DOT) with an immersed Clark-type electrode in the bulk liquid of the flask. Veljkovic et al. [65] used the sulfite system to investigate mass transfer coefficients in shake flasks. Maier and Büchs [31] used a 1-M sodium sulfite system, catalyzed by cobalt sulfate, to investigate the maximum oxygen transfer capacity of shake flasks. To measure the maximum oxygen transfer rate (OTR), they used a respiration activity monitoring system (RAMOS, described in Section 2.17.9.1). Duetz and Witholt [15] described a method to measure the OTR using a combination of glucose oxidase and horseradish peroxidase. Ortiz-Ochoa et al. [40] described the application of a catechol-2,3-dioxygenase for the bio-oxidation of catechol to determine oxygen transfer coefficients in small-scale vessels. In general, a chemical model system has many advantages for the investigation of the maximum oxygen transfer capacity of shake flasks. A chemical system is easy to handle, does not require sterile conditions, and allows the adjustment of the oxygen consumption by adding different catalyst concentrations. Furthermore, with a chemical oxygen consumer it can be assured that oxygen-limited conditions are prevailing.

2.17.5.2

Influence of Flask Wall Material on Liquid Film and Oxygen Transfer

Maier and Büchs [31] developed a physical model for the systematic investigation of the maximum oxygen transfer capacity of shake flasks. Two quite different effects contribute to the total oxygen mass transfer in hydrophilic glass flasks. One effect is the direct transfer from the gas phase into the rotating bulk liquid. In addition, the rotating bulk liquid deposits a thin liquid film on the hydrophilic glass wall and on the bottom of the flask. This liquid film also absorbs oxygen. According to the model, the mass transfer via the liquid film has a considerable contribution to the total mass transfer. Calculated values using the physical model and measured values of the maximum oxygen transfer capacity show a good agreement within a deviation of 20%. Experiments with hydrophobic shake flasks, where no liquid film is prevailing, showed significant lower mass transfer capacities, compared to hydrophilic flasks [31]. Figure 3 shows the difference of the maximum oxygen transfer capacity of hydrophilic and hydrophobic shake flasks.

2.17.5.3

Influence of Operation Conditions on Oxygen Transfer

The oxygen transfer from a gas phase to a liquid phase can generally be described as follows:   OTR ¼ kL a⋅LO2 ⋅pabs ⋅ yO2 ; headspace −yO2 ; liquid

½19

Shake-Flask Bioreactors

219

Maximum oxygen transfer capacity (OTRmax) (mmol l–1 h–1)

20

15

Normal hydrophilic

flask

10

5 Hydrophobic flask

0 50

100

150

200

250

300

Shaking frequency (n) (min−1) Figure 3 Influence of hydrophilic or hydrophobic surface properties of the flask wall on the maximum oxygen transfer capacity. Measurements conducted in 250-ml flask at 5-cm shaking diameter and 26-ml filling volume.

with the mass transfer coefficient kL , the specific interfacial area a, the oxygen solubility LO2 , the absolute pressure pabs, the mole fraction of oxygen in the head space yO2 ; headspace , and the mole fraction of oxygen in the gas phase yO2 ; liquid . In contrast to stirredtank fermenter, in shake flasks the specific interfacial area a is not effected by surfactants or coalescence. The values of the mass transfer coefficient kL and the volume-specific interfacial area a depend on the operating conditions and the properties of the biological or chemical system. In addition to the above-mentioned physical model, the following empirical correlation for kL a values of a sulfite system was determined [32, 57], considering the influence of the shaking frequency n, the filling volume VL, the shaking diameter d0, and the maximum inner flask diameter d: kL asulfit ¼ 6:67 ⋅10 − 6 ⋅ n1:16 ⋅ VL−0:83 ⋅ d00:38 ⋅ d1:92

½20

According to the above equation, the kL a, and thus the maximum oxygen transfer capacity, is increasing with increasing shaking frequency, shaking diameter, and flask diameter. In contrast to that, the kL a is decreasing with increasing filling volume. The kL a values according to eqn 20 have to be multiplied by a factor of 1–2.8 in case of fermentation systems as the oxygen solubility and diffusivity are specific to the culture media [1, 33, 57]. Sufficient oxygen transfer and mixing can also be achieved in unbaffled flasks. Baffles are not absolutely imperative. However, actively breathing microorganisms such as Escherichia coli require the use of small filling volumes (e.g., 10 ml liquid volume in a 250-ml flask) and relative high shaking frequencies (up to 400 rpm). At these conditions, a proper balancing combined with a permanent constant load of the shaker tray are important issues to assure smooth operation of the shaker without strong vibrations. However, most currently available commercial shakers are not designed to run at these high shaking frequencies (>300 rpm) for an extended time. To assure a sufficient oxygen supply of microorganisms, such as yeast or bacteria, it is strongly recommended to invest in high-performance shakers, which allow the change of the shaking diameter and balancing of the machine in accordance to the weight load on the shaker trays, to allow a continuous operation up to 400 rpm.

2.17.5.4

Mass Transfer Resistance of Sterile Plugs

The overall maximum oxygen transfer capacity of shake flasks depends on the oxygen transfer through the shake-flask closure, as for example, cotton, paper, or polymer foam plug, and the oxygen transfer through the gas/liquid interface [37, 39, 55]. For usual cultivation conditions, the oxygen transfer resistance of the flask closure is much smaller than the oxygen transfer resistance of the gas/liquid interface [17]. Consequently, the transfer resistance of the flask closure can only become relevant for very high values of the OTR, for example, as they can be obtained with baffled shake flasks or at high shaking frequencies [37]. The first systematic investigation to determine the diffusion coefficient of cotton plugs with different densities is reported by Schultz [55]. Gupta and Rao [17] used a dissolved oxygen fluorescence probe to estimate the mass transfer resistance of shake flasks. Henzler et al. [18] described a method to determine the diffusion coefficient of cotton plugs for carbon dioxide and oxygen. In 1991, Henzler and Schedel presented an extended model for the determination of diffusion coefficients of cotton plugs, considering Stefan flow. Mrotzek et al. [37] and Anderlei et al. [3] reported a new and very easy to use evaluation method for the exact determination of the

220

Bioreactors – Design

Air

Diffusion coefficient of carbon dioxide (cm2 s–1)

0.15

0.10

H

0.05

0 0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Bulk density of the plug (g cm–3) Figure 4 Diffusion coefficient for carbon dioxide over the bulk density of the cotton plug for shake flasks with a nominal volume from 50 to 5000 ml. The results are independent of the shape of the cotton plugs. Only the height H of the plug is put into the flask neck is relevant.

carbon dioxide diffusion coefficient, by measuring the water evaporation in shake flasks simply by using a balance. The diffusion coefficient for carbon dioxide is specified, as this parameter is independent of the gas concentration in the plug unlike the diffusion coefficient of oxygen [18]. Aluminum caps as flask closure are not recommended as it was shown that these sterile barriers allow a convective flow of gas into the flask [37]. This may cause contamination problems during the cultivation. Figure 4 shows different values for the diffusion coefficient of carbon dioxide in shake flasks for different bulk densities of the cotton plug [37]. Glass fiber and paper plugs can be treated like cotton plugs. For a rough estimation of the mass transfer through a plug of a shake-flask, eqn 21 can be applied: OTR ¼

 kpl  ⋅ yO2 ; air − yO2 ; headspace VL

½21

with the mass transfer coefficient of the plug kpl, the liquid filling volume VL , the mole fraction of oxygen outside of the flask yO2 ; air , and in the headspace of the flask yO2 ; headspace . The mass transfer resistance kpl can be described by: kpl ≈

DO2 ; eff ⋅Apl Hpl ⋅Vmo

½22

with the diffusion coefficient of oxygen in the plug material DO2 ; eff , the cross-section area Apl and the height Hpl of the plug, and the molar gas volume Vmo (Vmo = 22.414 l mol−1). Equations 21 and 22 can be combined with eqn 19 to yield the overall OTR:   kL a ⋅pabs ⋅LO2 yO2 ; headspace − yO2 ; liquid OTR ¼ ½23 k a⋅p ⋅L ⋅V 1+ L abskpl O2 L Equation 20 can be used in combination with an appropriate proportionality factor for the determination of the kL a value in eqn 23.

2.17.6 Baffled Shake Flasks Baffled shake flasks are mainly used to reach higher OTRs and enhanced mixing capacities at equivalent shaking frequencies compared to unbaffled flasks. However, whether this strategy is successful or not depends on the operating conditions (in-phase conditions). Despite of the above-mentioned advantages, many papers advise not to use baffled shake flasks [10, 19, 34]. Experiments in baffled flask are in general less reproducible compared to experiments in unbaffled flasks because of several reasons. McDaniel et al. [34] already reported the higher statistical deviation of measured data obtained from experiments in baffled flasks compared to data obtained in unbaffled flasks. Baffled flasks are prevalently custom made in local glass blower workshops and individually designed. Their geometry is difficult to reproduce, which makes it quite difficult to investigate general valid engineering characteristics [10]. Furthermore, the splashing liquid in baffled flasks might cause a wetting of the cotton plug and, thereby, lead to an impaired oxygen supply of the culture [19].

2.17.6.1

Specific Power Input in Baffled Shake Flasks

Up to now, only very few measurements of power input in baffled shake flasks have been reported [59]. The volumetric power consumption in baffled flasks was found to be significantly higher than in unbaffled flasks, as long as the fluid motion is in phase. As shown in Figure 5, the dimensionless Newton number is independent of the Reynolds number [48]. Thus, the power input

Shake-Flask Bioreactors

221

Modified newton number Ne′ (–)

101

100

Unbaffled flasks 10–1 4 10

Reynolds number Re (–)

105

Figure 5 Plot of the modified Newton number (Ne′) on the flask Reynolds number (Re) for baffled shake flasks. Measurements were conducted at ‘in-phase’ operation conditions with deionized water. Flask size, shaking diameter, and filling volume are labeled as follows: () VK = 500 ml, d0 = 100 mm, VL = 100 ml; (*) VK = 500 ml, d0 = 100 mm, VL = 50 ml; (+) VK = 500 ml, d0 = 100 mm, VL = 30 ml, (•) VK = 300 ml, d0 = 100 mm, VL = 60 ml; (▴) VK = 300 ml, d0 = 70 mm, VL = 60 ml; (♦) VK = 300 ml, d0 = 50 mm, VL = 60 ml; (•) VK = 300 ml, d0 = 100 mm, VL = 30 ml; (▴) VK = 300 ml, d0 = 70 mm, VL = 30 ml; (♦) VK = 300 ml, d0 = 50 mm, VL = 30 ml; (○) VK = 300 ml, d0 = 100 mm, VL = 15 ml; ( ) VK = 300 ml, d0 = 70 mm, VL = 15 ml; (◊) VK = 300 ml, d0 = 50 mm, VL = 15 ml; (−) unbaffled shake flasks according to eqn 1.2.10.

81.3 mm

35 mm

65 mm

▵

101 mm

20 mm

20 mm

120°

120°

14 mm

25 mm 300- ml flask

500- ml flask

Figure 6 Geometry of the baffled shake flasks used for the power input measurements.

characteristic of baffled flasks is qualitatively similar to the power input characteristic of stirred-tank reactors. The geometry of the shake flasks used for the specific power input measurements is shown in Figure 6. However, up to now, no concise and conclusive correlation of power input in baffled flasks, considering all possible geometric variations, has been published.

2.17.6.2

Out-of-Phase Phenomena in Baffled Shake Flasks

In unbaffled shake flasks, with a nominal volume of ≤1 l, out-of-phase conditions will only occur at elevated viscosities. In contrast, in baffled shake flasks, out-of-phase operating conditions were also registered for liquids with water-like viscosities. Baffles that are too large in size and number will decelerate the rotational movement of the bulk liquid similar to elevated viscosity [11]. In that case, a part of the liquid remains at the flask bottom and power input, mixing, and oxygen transfer is reduced. Therefore, if baffled flasks are used, baffle geometry and number of baffles as well as operating conditions must be carefully designed and balanced to obtain an intensification of hydrodynamics and not the opposite compared to unbaffled flasks [10].

2.17.6.3

Maximum Energy Dissipation Rate in Baffled Shake Flasks

For the calculation of the maximum energy dissipation rate (εmax) for baffled shake flasks, Peter et al. [47] recommended to use eqn 15 for all Reynolds numbers.

222

2.17.6.4

Bioreactors – Design Oxygen Transfer in Baffled Shake Flasks

The maximum oxygen transfer capacity in baffled shake flasks can be noticeable higher than in unbaffled flasks [17, 19]. Especially for high filling volumes, the increased gas–liquid interfacial area in baffled shake flasks leads to an increased maximum oxygen transfer capacity. However, the spraying of liquid that usually occurs for high filling volumes in baffled flasks could moisten the cotton closure of the flask [19]. This could lead to a higher oxygen transfer resistance of the cotton plug and to an increased risk of contamination. Therefore, the shaking frequency in baffled flasks must be limited to prevent a wetting of the cotton plug. The maximum oxygen transfer capacity in unbaffled flasks at high shaking frequencies is not lower than in baffled flasks, considering the lower limit for the maximum shaking frequency in baffled shake flasks [19]. To this date, no mathematical correlation is available to calculate the maximum OTR in baffled shake flasks (as a function of baffle geometry).

2.17.7 Use of Engineering Parameters for Scale-Up from Shake Flask to Stirred-Tank Reactor Experiments in shake flasks are often conducted with the intention to transfer process conditions to a larger reactor system after optimal cultivation conditions are found. Different culture conditions require different scale-up strategies [43, 60]. In the following, some examples of successful scale-up strategies are reported.

2.17.7.1

Specific Power Input as Scale-Up Parameter

The specific power input is one of the crucial parameters for the scale-up of bioprocesses. An equal specific power input leads to similar flow conditions in geometrically similar reactor systems of different sizes. Power input per volume can be easily measured, in contrast to parameters that are usually used to describe fluid flow characteristics, as for example, the local fluid flow velocity or the shear rate. Reyes et al. [50] used initial power input values to transfer cultivation conditions of Azotobacter vinelandii culture, producing the biopolymer alginate, from shake flasks to a stirred-tank reactor. Despite equal power input values at the beginning of the fermentation, the polymer obtained from the cultivation in a stirred-tank reactor had a lower mean molecular mass compared to the polymer produced in shake flasks. The reason for this was shown to be the strongly increasing viscosity of the fermentation broth during the cultivation. As a result, the specific power input in shake flasks also increased during the fermentation as the modified Newton number increases with decreasing Reynolds number (see Figure 2). In contrast, the Newton number in stirred-tank reactors in the turbulent regime is independent of the Reynolds number and viscosity. Therefore, the progress of specific power input in shake flasks is different compared to that in a stirred-tank reactor [50]. Pena et al. [44] measured the progress of the specific power input during the cultivation of A. vinelandii in shake flasks and simulated the specific power input profile in a stirred-tank reactor. With this method, it was possible to produce alginate with a similar mean molecular mass in a stirred-tank reactor, compared to the alginate produced in shake flasks [44]. Mehmood et al. investigated the pristinamycin formation with Streptomyces pristinaespiralis. They could nicely correlate their results from shake flasks of different size on the specific power input, calculated with eqn 10. They observed a clear optimum of product formation at a specific power input value of about 5 kW m−3. However, the growth of their microorganisms could be better described as a function of kL a values, which were estimated using eqn 20.

2.17.7.2

Maximum Energy Dissipation Rate as Scale-Up Parameter

Takebe et al. [61] were able to correlate their results on Neurospora crassa fermentations in unbaffled and partially baffled flasks and in a 30-l jar fermenter with what they termed ‘agitation intensity’. The agitation intensity represented drop-size measurements of a model liquid two-phase system. As drop dispersion is governed by hydromechanical stress, the results of Takebe et al. [61] have to be regarded as an early example of successful correlation of fermentation data of a filamentous fungi as a function of hydro­ mechanical stress.

2.17.7.3

Scale-Up of Ventilation from Shake Flask to Stirred-Tank Reactor

A sufficient oxygen supply is a necessary requirement for optimal growth and product formation of many aerobic micro­ organisms. In addition, the concentration of carbon dioxide in the fermentation broth is important for some bioprocesses [26, 56] . An estimation of the required specific gas flow rate of a stirred reactor, for achieving similar head space concentra­ tions as in shake flasks, is presented in Figure 7. The values for the recommended gas flow rates in a fermenter are plotted against the values of the used liquid filling volume in shake flasks. As an example, if fermentation in a 250-ml narrow neck shake flask was successfully performed with 25 ml filling volume, the optimal specific gas flow rate in a fermenter would be 0.5 vvm in order to obtain the same level of ventilation in both types of bioreactors. These can only be regarded as rough estimates. For a detailed match of ventilation, unsteady-state conditions have to be considered for the head space as shown by Amoabediny and Buechs (2007).

Shake-Flask Bioreactors

223

3

Specific aeration rate (vvm)

2.5

250, 500, 1000 ml wide neck, 2000 ml narrow neck 50, 100 ml wide neck, 250, 500 ml narrow neck

2

1.5

Valid for: 0 < RQ < 1.5 (–) 0 < OTR < 0.1 (mol l–1 h–1)

1

0.5

0 0

10

20

30

40

50

60

70

80

90

100

110

Filling volume (ml) Figure 7 Plot of the specific gas flow rate in a fermenter against the used liquid filling volume in shake flask to obtain the same ventilation in both fermentation systems. The error bars indicate the variations that result from changing oxygen transfer rates and respiration RQs. Calculated with the model described in Mrotzek et al. [37].

2.17.8 Fed-Batch and Continuous Cultures in Shake Flasks Currently, screening cultures in shake flasks are mostly performed in batch mode, whereas bench- and technical-scale fermentations are usually performed in fed-batch mode. This is due to the fact that most microorganisms applied in fermentation processes show an overflow metabolism at elevated concentration of the carbon source. In addition, often an inhibition by high substrate or osmotic pressure is observed. The production of most secondary metabolites is catabolite repressed. In this case, the disadvantage of batch in comparison to fed-batch operation mode is particularly high. Weuster-Botz et al. [66] have reported on a system with 16 shake flasks where a precise syringe pump distributes nutrient solution via a multiplexing system and tubes to the different bioreactors. Jeude et al. [23] have developed a polymer-based controlled release system, which consists of a silicon matrix in which glucose is embedded. From this matrix, glucose is released with a predetermined kinetics as soon as it gets into contact with an aqueous solution. The release kinetics were adjusted to the nutrient requirement of bacteria and yeast. With this very-easy-to-handle system, a fed-batch culture mode was realized. Jeude et al. [23] could demonstrate that E. coli and Hansenula polymorpha cultures showed completely different production characteristics in batch and fed-batch modes. In some examples, a 40–400-fold increase of product formation was observed. Increased product formation was also obtained by Panula-Perala et al. [42] in their work. They developed a system using the enzymatic hydrolysis of starch by glucoamylase to realize a glucose-limited fed-batch fermentation. Stöckmann et al. [58] and Scheidle et al. [53] have shown for H. polymorpha that the ranking of the selected strains change if batch versus fed-batch operation mode is used during screening. Therefore, the optimal strains for later production in large fed-batch fermentations were not found in the batch screenings. Fed-batch fermentation mode applying controlled release systems was also used to synchronize and equalize growth in parallel precultures [22] in order to ensure equivalent starting conditions for different main cultures.

2.17.9 Online Measuring Techniques in Shake Flasks A detailed knowledge about cultivation conditions is essential for systematic screening experiments. Parameters such as the pH value and the temperature of the fermentation broth as well as DOT and the OTR of the culture are important for optimization of culture conditions and scale-up.

2.17.9.1

Online Measuring Techniques of the OTR and Carbon Dioxide Transfer Rate

Most metabolic activities of aerobic microorganisms are associated with oxygen consumption of the culture. Different studies deal with the oxygen supply of microbial cultures in shake flasks [35, 56, Gaden, 1962]. A noninvasive measuring technique, the RAMOS, for the online determination of the OTR, the carbon dioxide transfer rate, and the respiratory quotient (RQ) in shake flasks, was developed by Anderlei and Büchs [5] and Anderlei et al. [4]. The device is equipped with eight

224

Bioreactors – Design

Cotton plug

O2 Sensor

Gas

inlet

Equal gas head space concentration

Gas outlet

Normal flask

RAMOS flask Equal liquid hydrodynamics Figure 8 Modified shake flasks for the respiration activity monitoring system (RAMOS).

modified shake flasks (Figure 8). An oxygen partial pressure sensor is mounted on top of each flask and three ports for gas inlet, outlet, and inoculation are mounted on each flask. A pressure sensor enables the determination of the differential gas pressure in the head space of the flask and, therefore, the RQ. The connections for gas inlet and outlet are equipped with valves, to control the air flow through the flask. During fermentation, a measuring cycle with a rinsing phase (where the valves are opened) and a measuring phase (where the valves are closed) is continuously repeated [4]. During the measuring phase, the respiration of the microorganisms leads to a decrease of oxygen partial pressure in the head space of the flask. A commercial version of the device is available from Kühner AG, Birsfelden, Swiss and Hitec Zang GmbH, Herzogenrath, Germany. Another system to measure the gas transfer rates was developed by the company BlueSens GmbH, Herten, Germany. Their device measures oxygen and carbon dioxide in the headspace of a shake flask. The minimal nominal volume of the flasks for which this technique can be applied is 500 ml. A special sterile closure has to be used for the measurement. Figure 9 shows a measurement example obtained by the RAMOS device. In this experiment, eight flasks of the system were filled with medium of different phosphate concentration in order to determine the minimum amount of phosphate requirement by the investigated E. coli strain. All cultures initially start to grow exponentially. As soon as phosphate limitation occurs, a sharp transition of the breathing activity into a slowly decreasing trend is observed. Subsequently, the breathing activity decreases abruptly, which is due to the glucose exhaustion (e.g., for the culture with 0.2 g l−1 phosphate at about 15 h). In the next phase, the culture is metabolizing acetate which has been formed during the first phase as overflow metabolite from glucose. Finally, the breathing drops down to nearly zero. From the results, it becomes obvious that the culture is slightly limited at a phosphate concentration of 0.4 g l−1 but not at 0.5 g l−1. This example demonstrates that with systematic multiple parallel culture experiments, it is possible to estimate medium requirements of microorganisms without taking any sample and without the necessity of performing offline analysis.

2.17.9.2

Online Measurement of the DOT

Common methods to investigate DOT require invasive Clark-type electrodes that are, for example, used in stirred-tank fermenters [51, 64]. However, these relatively large electrodes act like baffles and change the hydrodynamic conditions in comparison to shake flasks without DOT measurement. Tolosa et al. [62] and Wittmann et al. [67] presented noninvasive measuring techniques for determining the DOT in shake flasks. These methods apply oxygen-sensitive fluorescence dyes immobilized in an autoclavable patch, attached on the flask bottom. Light-emitting diodes are used for excitation of the fluorophore and the emission is analyzed via a detector. These techniques are now commercially available from PreSens GmbH, Regensburg, Germany and Fluorometrix Corp, Baltimore, USA. Gupta and Rao [17] reported the successful application of this method to examine the DOT of shake flasks using a relative filling volume of 40%. The high filling volume is necessary to ensure that the patch is permanently covered by liquid [17]. Consequently, a direct contact between the gas phase and the oxygen-sensitive patch may lead to wrong measurements.

2.17.9.3

Online Measurement of the pH Value

Similar to the DOT, pH values were initially measured in shake flasks by ordinary pH glass electrodes [63, 66] immersed into the liquid. During the last years, fluorescence optodes were developed [6, 24, 28], which do not interfere with the hydrodynamics in the flasks. Scheidle et al. [52] reported on the utilization of these techniques for the measurement of the pH value in a microbial culture in shake flasks.

Shake-Flask Bioreactors

225

0.04

Oxygen transfer rate (OTR) (mol l–1 h–1)

1 and 0.5 g l–1

0.035 0.4 g l–1

0.03 0.025 0.3 g l–1

0.02 0.2 g l–1

0.015 0 g l–1

0.01

0.1 g l–1

0.005 0.05 g l–1

0 0

5

10

15

20 Time (h)

25

30

35

Figure 9 Determination of the minimum amount of phosphate required by E. coli BL 21 (DE3); 250-ml flask, 12.5-ml WR mineral medium with 100-mM MOPS buffer, 30 °C, 300 rpm, 5-cm shaking diameter.

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