Maximum Local Energy Dissipation Research Articles

Impact of hydromechanical stress on CHO cells’ metabolism and productivity: Insights from shake flask cultivations with online monitoring of the respiration activity

The hydromechanical stress is a relevant parameter for mammalian cell cultivations, especially regarding scale-up processes. It describes the mechanical forces exerted on cells in a bioreactor. The maximum local energy dissipation rate is a suitable parameter to characterize hydromechanical stress. In literature, different studies deal with the effects of hydromechanical stress on CHO cells in stirred tank reactors. However, they often focus on lethal effects. Furthermore, systematic examinations in smaller scales like shake flasks are missing. Thus, this study systematically considers the influence of hydromechanical stress on CHO DP12 cells in shake flask cultivations. By utilizing online monitoring of the oxygen transfer rate, the study simplifies and enhances the resolution of examinations. Results indicate that while lethal effects are absent, numerous sub-lethal effects emerge with increasing hydromechanical stress: The process time is prolonged. The time of glucose and glutamine depletion, and the lactate switch correlate positively linear with the logarithmic average energy dissipation rate while the maximum specific growth rate correlates negatively. Strikingly, the final antibody concentration only declines at the highest tested average energy dissipation rate of 3.84Wkg-1 (only tested condition with a turbulent flow regime and therefore a higher maximal local energy dissipation rate) from about 250mgL-1 to about 180mgL-1. This study presents a straightforward method to examine the impact of hydromechanical stress in shake flasks, easily applicable to any other suspension cell line. Additionally, it offers valuable insights for scale-up processes, for example into stirred tank reactors.

Scale-up of an amoeba-based process for the production of the cannabinoid precursor olivetolic acid

BackgroundThe availability of new biological platform organisms to get access to innovative products and processes is fundamental for the progress in biotechnology and bioeconomy. The amoeba Dictyostelium discoideum represents a novel host system that has recently been employed for both the discovery of new natural products and as a cell factory for the production of bioactive compounds such as phytochemicals. However, an essential parameter to evaluate the potential of a new host system is the demonstration of its scalability to allow industrial applicability. Here, we aimed to develop a bioprocess for the production of olivetolic acid, the main precursor of cannabinoids synthesized by a recently engineered D. discoideum strain.ResultsIn this study, a sophisticated approach is described to scale-up an amoeba-based polyketide production process in stirred tank bioreactors. Due to the shear sensitivity of the cell wall lacking amoebae, the maximum local energy dissipation rate (εmax) was selected as a measure for the hydromechanical stress level among different scales. By performing 1.6-L scale batch fermentations with different stress conditions, we determined a maximum tolerable εmax of 3.9 W/kg for D. discoideum. Further, we used this parameter as scale-up criterion to develop a bioprocess for olivetolic acid production starting from a 7-L stirred tank reactor to the industrially relevant 300-L scale with a product concentration of 4.8 µg/L, a productivity of 0.04 µg/L/h and a yield of 0.56 µg/g glucose.ConclusionWe developed a robust and reliable scale-up strategy for amoeba-based bioprocesses and evaluated its applicability for the production of the cannabinoid precursor olivetolic acid. By determining the maximum tolerable hydromechanical stress level for D. discoideum, we were able to scale-up the process from shake flasks to the 300-L stirred tank reactor without any yield reduction from cell shearing. Hence, we showed the scalability and biotechnological exploitation of amoeba-based processes that can provide a reasonable alternative to chemical syntheses or extractions of phytochemicals from plant biomass.

Stability of polymersomes with focus on their use as nanoreactors.

The increased membrane stability of polymersomes compared to their liposomal counterparts is one of their most important advantages. Due to this benefit, polymer vesicles are intended to be used not only as carrier systems for drug delivery purposes but also as nanoreactors for biotechnological applications. Within this work, the stability of polymersomes made of the triblock copolymer poly(2-methyloxazoline)15-poly(dimethylsiloxane)68-poly(2-methyloxazoline)15 (PMOXA15-PDMS68-PMOXA15) toward mechanical stress, typically prevailing in stirred-tank reactors being the most often used reactor type in the biotechnological industry, was characterized. Dynamic light scattering and turbidity measurements showed that stirrer rotation causing a maximum local energy dissipation of up to 1.23W/kg-1 did not result in any loss of vesicle quality or quantity. Nevertheless, most probably due to local membrane defects, 6.6% release of the previously encapsulated model dye calcein was recognized at 25°C within 48h. Moreover, increased temperature, leading to decreased membrane viscosity and increased membrane fluidity, respectively, led to a higher molecule leakage. Besides, the stability of polymersomes in two-phase systems was investigated. Although alkanes and ionic liquids were shown not to lead to complete vesicle damage, no efficient calcein retention was achieved in either case.

Large-scale crystallization of proteins for purification and formulation.

Since about 170years, salts were used to create supersaturated solutions and crystallize proteins. The dehydrating effect of salts as well as their kosmotropic or chaotropic character was revealed. Even the suitability of organic solvents for crystallization was already recognized. Interestingly, what was performed during the early times is still practiced today. A lot of effort was put into understanding the underlying physico-chemical interaction mechanisms leading to protein crystallization. However, it was understood that already the solvation of proteins is a highly complex process not to mention the intricate interrelation of electrostatic and hydrophobic interactions taking place. Although many basic questions are still unanswered, preparative protein crystallization was attempted as illustrated in the presented case studies. Due to the highly variable nature of crystallization, individual design of the crystallization process is needed in every single case. It was shown that preparative crystallization from impure protein solutions as a capture step is possible after applying adequate pre-treatment procedures like precipitation or extraction. Protein crystallization can replace one or more chromatography steps. It was further shown that crystallization can serve as an attractive alternative means for formulation of therapeutic proteins. Crystalline proteins can offer enhanced purity and enable highly concentrated doses of the active ingredient. Easy scalability of the proposed protein crystallization processes was shown using the maximum local energy dissipation as a suitable scale-up criterion. Molecular modeling and target-oriented protein engineering may allow protein crystallization to become part of a platform purification process in the near future.

Characterization of hydromechanical stress in aerated stirred tanks up to 40 m(3) scale by measurement of maximum stable drop size.

BackgroundTurbulence intensity, or hydromechanical stress, is a parameter that influences a broad range of processes in the fields of chemical engineering and biotechnology. Fermentation processes are often characterized by high agitation and aeration intensity resulting in high gas void fractions of up to 20% in large scale reactors. Very little experimental data on hydromechanical stress for such operating conditions exists because of the problems associated with measuring hydromechanical stress under aeration and intense agitation.ResultsAn indirect method to quantify hydromechanical stress for aerated operating conditions by the measurement of maximum stable drop size in a break-up controlled dispersion was applied to characterize hydromechanical stress in reactor scales of 50 L, 3 m3 and 40 m3 volume with a broad range of operating conditions and impeller geometries (Rushton turbines). Results for impellers within each scale for the ratio of maximum to specific energy dissipation rate ϕ based on measured values of maximum stable drop size for aerated operating conditions are qualitatively in agreement with results from literature correlations for unaerated operating conditions. Comparison of data in the different scales shows that there is a scale effect that results in higher values for ϕ in larger reactors. This behavior is not covered by the classic theory of turbulent drop dispersion but is in good agreement with the theory of turbulence intermittency. The data for all impeller configurations and all aeration rates for the three scales can be correlated within ±20% when calculated values for ϕ based on the measured values for dmax are used to calculate the maximum local energy dissipation rate. A correlation of the data for all scales and all impeller configurations in the form ϕ = 2.3∙(ϕunaerated)0.34∙(DR)0.543 is suggested that successfully models the influence of scale and impeller geometry on ϕ for aerated operating conditions.ConclusionsThe results show that besides the impeller geometry, also aeration and scale strongly influence hydromechanical stress. Incorporating these effects is beneficial for a successful scale up or scale down of this parameter. This can be done by applying the suggested correlation or by measuring hydromechanical stress with the experimental method used in this study.

Maximum stable drop size measurements indicate turbulence attenuation by aeration in a 3 m3 aerated stirred tank

In this study, break-up controlled drop dispersion experiments in a 3m3 pilot scale reactor with 1.2m inner diameter are presented that allow a correlation of the maximum local energy dissipation rate in stirred reactors with intense aeration. Experiments with two different setups of 6-bladed Rushton turbine impellers with diameters of d=0.41m (d/DR=0.34) and d=0.51m (d/DR=0.43) in a 3 impeller configuration were conducted. The results from experiments without aeration are well in agreement with the existing literature on drop dispersion. The results from experiments with aeration indicate a strong attenuation of turbulence intensity in stirred tank reactors by the presence of air. The ratio between maximum and volume-averaged energy dissipation rate was reduced by aeration by 64% for the d/DR=0.34 impeller and by 52% for the d/DR=0.43 impeller when compared with unaerated operating conditions on the basis of equal volumetric power input. The value of the aeration rate had no measurable effect in the range of aeration rates applied, which was between 0.1vvm (volume of gas/volume liquid/minute) and 1vvm.

Protein crystallization in stirred systems—scale‐up via the maximum local energy dissipation

Macromolecular bioproducts like therapeutic proteins have usually been crystallized with µL-scale vapor diffusion experiments for structure determination by X-ray diffraction. Little systematic know-how exists for technical-scale protein crystallization in stirred vessels. In this study, the Fab-fragment of the therapeutic antibody Canakinumab was successfully crystallized in a stirred-tank reactor on a 6 mL-scale. A four times faster onset of crystallization of the Fab-fragment was observed compared to the non-agitated 10 µL-scale. Further studies on a liter-scale with lysozyme confirmed this effect. A 10 times faster onset of crystallization was observed in this case at an optimum stirrer speed. Commonly suggested scale-up criteria (i.e., minimum stirrer speed to keep the protein crystals in suspension or constant impeller tip speed) were shown not to be successful. Therefore, the criterion of constant maximum local energy dissipation was applied for scale-up of the stirred crystallization process for the first time. The maximum local energy dissipation was estimated by measuring the drop size distribution of an oil/surfactant/water emulsion in stirred-tank reactors on a 6 mL-, 100 mL-, and 1 L-scale. A comparable crystallization behavior was achieved in all stirred-tank reactors when the maximum local energy dissipation was kept constant for scale-up. A maximum local energy dissipation of 2.2 W kg(-1) was identified to be the optimum for lysozyme crystallization at all scales under study.

DETERMINING COMPLETE SUSPENSION OF IMMOBILIZED ENZYMES BY ANALYSIS OF DIGITAL CAMERA IMAGES

To increase the stability of enzymes in organic solvents, they may be immobilized in a polymer matrix suspended in the solvent. It is important to know that all the immobilized enzyme particles are completely suspended in order to ensure an efficient particle-liquid mass transfer coefficient while putting as little energy into the system as necessary so that the particles are not harmed. A novel noninvasive method based on photometry and analysis of images taken with a standard digital camera is presented to estimate a mean extinction in different parts of the reactor and to finally determine the just suspending stirring speed (NJS) for complete suspension in an objective way. With this optical method, six different reactor configurations were compared based on the maximum local energy dissipation rate. Here, the 90° bladed stirrer with a flat bottom proved to be one of the best configurations for attaining complete suspension with low maximum local energy dissipation rate.

Evaluation of parallel milliliter-scale stirred-tank bioreactors for the study of biphasic whole-cell biocatalysis with ionic liquids

As clear structure-activity relationships are still rare for ionic liquids, preliminary experiments are necessary for the process development of biphasic whole-cell processes involving these solvents. To reduce the time investment and the material costs, the process development of such biphasic reaction systems would profit from a small-scale high-throughput platform. Exemplarily, the reduction of 2-octanone to (R)-2-octanol by a recombinant Escherichia coli in a biphasic ionic liquid/water system was studied in a miniaturized stirred-tank bioreactor system allowing the parallel operation of up to 48 reactors at the mL-scale. The results were compared to those obtained in a 20-fold larger stirred-tank reactor. The maximum local energy dissipation was evaluated at the larger scale and compared to the data available for the small-scale reactors, to verify if similar mass transfer could be obtained at both scales. Thereafter, the reaction kinetics and final conversions reached in different reactions setups were analysed. The results were in good agreement between both scales for varying ionic liquids and for ionic liquid volume fractions up to 40%. The parallel bioreactor system can thus be used for the process development of the majority of biphasic reaction systems involving ionic liquids, reducing the time and resource investment during the process development of this type of applications.

New milliliter‐scale stirred tank bioreactors for the cultivation of mycelium forming microorganisms

A novel milliliter-scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms on a 10 milliliter-scale. A newly designed one-sided paddle impeller is driven magnetically and rotates freely on an axis in an unbaffled reaction vessel made of polystyrene. A rotating lamella is formed which spreads out along the reactor wall. Thus an enhanced surface-to-volume ratio of the liquid phase is generated where oxygen is introduced via surface aeration. Volumetric oxygen transfer coefficients (k(L)a) > 0.15 s(-1) were measured. The fast moving liquid lamella efficiently prevents wall growth and foaming. Mean power consumption and maximum local energy dissipation were measured as function of operating conditions in the milliliter-scale stirred tank bioreactor (V = 10 mL) and compared to a standard laboratory-scale stirred tank bioreactor with six-bladed Rushton turbines (V = 2,000 mL). Mean power consumption increases with increasing impeller speed and shows the same characteristics and values on both scales. The maximum local energy dissipation of the milliliter-scale stirred tank bioreactor was reduced compared to the laboratory-scale at the same mean volumetric power input. Hence the milliliter impeller distributes power more uniformly in the reaction medium. Based on these data a reliable and robust scale-up of fermentation processes is possible. This was demonstrated with the cultivation of the actinomycete Streptomyces tendae on both scales. It was shown that the process performances were equivalent with regard to biomass concentration, mannitol consumption and production of the pharmaceutical relevant fungicide nikkomycin Z up to a process time of 120 h. A high parallel reproducibility was observed on the milliliter-scale (standard deviation < 8%) with up to 48 stirred tank bioreactors operated in a magnetic inductive drive. Rheological behavior of the culture broth was measured and showed a highly viscous shear-thinning non-Newtonian behavior. The newly developed one-sided paddle impellers operated in unbaffled reactors on a 10 milliliter-scale with a magnetic inductive drive for up to 48 parallel bioreactors allows for the first time the parallel bioprocess development with mycelium forming microorganisms. This is especially important since these kinds of cultivations normally exhibit process times of 100 h and more. Thus the operation of parallel stirred tank reactors will have the potential to reduce process development times drastically.

Power consumption and maximum energy dissipation in a milliliter‐scale bioreactor

Mean power consumption and maximum local energy dissipation were measured as function of operating conditions of a milliliter-scale stirred tank bioreactor (V = 12 mL) with a gas-inducing impeller. A standard laboratory-scale stirred tank bioreactor (V = 1,200 mL) with Rushton turbines was used as reference. The measured power characteristics (Newton number as function of Reynolds number) were the same on both scales. The changeover between laminar and turbulent flow regime was observed at a Reynolds number of 3,000 with the gas-inducing stirrer on a milliliter-scale. The Newton number (power number) in the turbulent flow regime was 3.3 on a milliliter-scale, which is close to values reported for six-blade Rushton turbines of standard bioreactors. Maximum local energy dissipation (epsilon(max)) was measured using a clay/polymer flocculation system. The maximum local energy dissipation in the milliliter-scale stirred tank bioreactor was reduced compared with the laboratory-scale stirred tank at the same mean power input per unit mass (epsilon(ø)), yielding epsilon(max)/epsilon(ø) approximately 10 compared with epsilon(max)/epsilon(ø) approximately 16. Hence, the milliliter-scale stirred tank reactor distributes power more uniformly in the reaction medium. These results are in good agreement with literature data, where a decreasing epsilon(max)/epsilon(ø) with increasing ratio of impeller diameter to reactor diameter is found (d/D = 0.7 compared with d/D = 0.4). Based on these data, impeller speeds can now be easily adjusted to achieve the same maximum local energy dissipation at different scales. This enables a more reliable and robust scale-up of bioprocesses from milliliter-scale to liter-scale reactors.

Hydromechanical stress in shake flasks: Correlation for the maximum local energy dissipation rate

Shake flasks are widely used in biotechnological process research. Bioprocesses for which hydromechanical stress may become the rate controlling parameter include those where oils are applied as carbon sources, biotransformation of compounds with low solubility in the aqueous phase, or processes employing animal, plant, or filamentous microorganisms. In this study, the maximum local energy dissipation rate as the measure for hydromechanical stress is characterized in shake flasks by measuring the maximum stable drop size. The theoretical basis for the method is that the maximum stable drop diameter in a coalescence inhibited liquid/liquid dispersion is only a function of the maximum local energy dissipation rate and not of the dispersing apparatus. The maximum local energy dissipation rate is obtained by comparing the drop diameters in shake flasks to those in a stirred tank reactor. At the same volumetric power consumption, the maximum energy dissipation rate in shake flasks is about 10 times lower than in stirred tank reactors explaining the common observation of considerable differences in the morphology of hydromechanically sensitive cells between these two reactor types. At the same volumetric power consumption, the maximum local energy dissipation rate in baffled and in unbaffled shake flasks is very similar. A correlation is presented to quantify the maximum local energy dissipation rate in shake flasks as a function of the operating conditions. Non-negligible drop viscosity may be considered by known literature correlations. Further, from dispersion experiments a critical Reynolds number of about 60,000 is proposed for turbulent flow in unbaffled shake flasks.

Study Of Micromixing in a Stirred Tank Using a Rushton Turbine: Comparison of Feed Positions and Other Mixing Devices

The consecutive-competing iodide–iodate reaction scheme has been used to study micromixing phenomena in a baffled vessel of 0.29 m diameter agitated by a Rushton turbine. It has been confirmed that, by using successive injections, this reaction scheme is very efficient for such a study. Four agitator speeds giving mean specific energy dissipation rates, e T from ∼0.2 to ∼ 1.2 Wkg −1 have been used, with sub-surface feeding at one of four points. For a given speed, addition at each of these four points gave different local values of e ¯ T, ranging from less than e ¯ T very close to the top of the liquid to much greater close to the impeller. The point closest to the impeller was chosen to be such that feeding was estimated to be at the point of maximum e T. For the maximum speed, the segregation index, as a measure of the amount of ’waste product’, was ∼20% with feed at the top of the liquid (as preferred industrially because of its convenience). This ’waste’ was reduced to ∼5% by feeding at the point of maximum e T close to the impeller. A comparison was also made with results reported in the literature using the same reaction for two new devices developed for improved micromixing. By feeding at the carefully chosen position close to the impeller, the results with the Rushton turbine were as good as or better than with the special devices even at the comparatively low e ¯ T of ∼ l.2 Wkg−1. It was estimated that the maximum local specific energy dissipation rate was ∼70 times the mean, in reasonable agreement with a very recent study where the same pair of reactions and LDV were both used.

A study of an up‐ and a down‐pumping wide‐blade hydrofoil impeller: Part II. CFD analysis

AbstractTwo different ways of using CFD to determine the flow field produced by the complex, wide‐blade hydrofoil APV‐B2 impeller are presented. The use of the “sliding mesh” approach was not successful and the reasons are discussed. Details of the numerical solution of the momentum transfer equations are described for the case of an axially‐symmetrical jet model. The flow field for the single impeller pumping either upwards or downwards at 200 rpm was simulated using the structured Fluent code. Good agreement of CFD predictions with LDA velocity data was found, though the maximum local energy dissipation rate was 2 1/2 to 5 times lower than was estimated from the experiments. The power numbers predicted by CFD were slightly higher than those obtained experimentally. The flow fields for dual and triple impellers pumping upwards were also simulated and used to guide larger scale homogenisation experiments.