Vergleich von Messungen des kLa-Wertes nach der stationären und der dynamischen Methode bei der Fermentation von L-Lysin und Turimycin

The calculation and scale-up of fermentation processes need k_La as one of the most important engineering data. There are two methods to determine k_La depending on power input, aeration rate and the properties of the fermentation broth: static with a balance between air supply and exit, dynamic gassing out with following the changes of dissolved oxygen concentration during periods of air off and a following air on. Within early intervals of fermentation time the data from both methods agree well, while for later time intervals the dynamic method always gives much lower values for k_La than static. The only explanations for this phenomenon are quick changes in the oxygen metabolism or an enzymatic storage of oxygen. For both gassing out and saturation period it is possible to calculate the same absolute amounts of this additional oxygen.     

K L a Evaluation during the course of fermentation using data reconciliation techniques

The oxygen mass transfer coefficient often serves to compare the efficiency of bioreactors and their mixing devices as well as being an important scale-up factor. In submerged fermentation, four methods are available to estimate the overall oxygen mass transfer coefficient (K_La): the dynamic method, the stationary method based on a previous determination of the oxygen uptake rate (Q_O2X), the gaseous oxygen balance and the carbon dioxide balance. Each method provides a distinct estimation of the value of K_La. Data reconciliation was used to obtain a more probable value of K_La during the production of Saccharomyces cerevisiae, performed in 22.5-l fed-batch bioreactor. The estimate of K_La is obtained by minimising an objective function that includes measurement terms and oxygen conservation models, each being weighted according to their level of confidence. Weighting factors of measurement terms were taken as their respective inverse variance whereas weighting factors of oxygen conservation models were obtained using Monte Carlo simulations. Results show that more coherent and precise estimations of K_La are obtained.     

Development of a scale-up strategy for Chinese hamster ovary cell culture processes using the kLa ratio as a direct indicator of gas stripping conditions

Herein, we described a scale-up strategy focused on the dissolved carbon dioxide concentration (dCO₂) during fed-batch cultivation of Chinese hamster ovary cells. A fed-batch culture process for a 2000-L scale stainless steel (SS) bioreactor was scaled-up from similarly shaped 200-L scale bioreactors based on power input per unit volume (P/V). However, during the 2000-L fed-batch culture, the dCO₂ was higher compared with the 200-L scale bioreactor. Therefore, we developed an alternative approach by evaluating the k_La values of O₂ (k_La[O₂]) and CO₂ [k_La(CO₂)] in the SS bioreactors as a scale-up factor for dCO₂ reduction. The k_La ratios [k_La(CO₂)/k_La(O₂)] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the k_La ratio of the 2000-L scale bioreactor became similar and the P/V value become smaller compared with those of the 200-L SS bioreactor. The dCO₂ trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This k_La ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The k_La ratios for the SUBs were evaluated and conditions that provided k_La ratios similar to the 200-L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the k_La ratio is a powerful scale-up factor useful to control dCO₂ during fed-batch cultures.     

用于红霉素生产的500m3生物反应器的理性设计

红霉素(erythromycin)是由绛红色糖多胞菌(Saccharopolyspora erythraea)发酵生产的次级代谢产物,其生产水平不仅受发酵工艺的影响,也受反应器结构影响。为解决红霉素发酵过程放大问题,本研究采用时间常数法和计算流体力学(computational fluid dynamics,CFD)数值模拟验证相结合的方法设计了500m³超大规模红霉素耗氧发酵生物反应器。首先,通过对50L反应器红霉素发酵过程研究,发现溶氧是关键性限制因素,通过氧消耗速率(oxygen uptake rate,OUR)等参数分析计算得到设备的氧供应时间常数t_mt需小于6.25s。然后,基于时间常数法和经验关联式理性设计500m³反应器搅拌桨叶组合方式,即底层BDT8桨叶+两层MSX4桨叶的搅拌桨组合,并通过经验公式及CFD方法对设计结果进行了模拟验证。两种验证方法结果均表明500m³反应器采取底层BDT8桨叶+两层MSX4桨叶的组合方式时设备的氧供应时间常数小于6.25s,且反应器内流场特性(如持气率、剪切率和速度矢量等)均能满足红霉素大规模发酵的需要。经实际发酵验证,设计的生物反应器能够满足红霉素的工业规模发酵应用。     

Oxygen transfer in Streptomyces fermentation broths

The oxygen transfer coefficient has been investigated in S. noursci and S. lavendulae fermentation broths obtained from fermentors of different operating volumes (61., 30001., 20,0001.). Fermentors had K_La_s values ranging from 1.0 to 17.0 min^?1, calculated from sulphite oxidation rates. The dynamic measurement of the volumetric oxygen transfer coefficient. (K_La) has been performed in the different fermenting systems. As the fermentation progressed, especially in the first stages, K_La values have decreased in both fermentations and in each system of fermentors. In order to characterise the whole fermenting system an average K_La_s was calculated from the obtained K_La values. The average K_La grew with increasing K_La_s values and ranged from 0.03 to 3.72 min^?l. Some factors possibly having an influence on the, change of K_La have been studied. The oxygen transfer coefficients of the broths have been measured in falling films and ranged from 0.05 to 0.4 cm min^?1. The flow conditions have been characterized by Reynolds numbers of broths varying between 1.0 and 60.0. The average thickness of the falling films have been measured and plotted against Reynolds number. The Re⁺ which is the breaking-point of the plot increased as the fermentation proceeded. In the region of Re⁺ the values of the oxygen transfer coefficient increased rapidly. An approximate correlation could be established between the Re⁺ and the physical properties of fermentation broth.     

Oxygen transfer coefficients by dynamic model moment analysis

A new method to estimate the oxygen transfer coefficient (K_La) from the experimental dynamic response data is presented. Employing a linear model which allows for gas phase, diffusion film, and oxygen electrode dynamics, the first moment of the response curve is simply related to the sum of the model parameters. Two separate experiments are used to characterize the measurement dynamics and to measure the unknown K_La parameter. The simple calculation procedure involves only measuring the area above the response curves.     

Oxygen transfer to mixed cultures in tower systems

A modified dynamic method is introduced to determine the oxygen transfer coefficient, K_L a, in aerobic fermentation systems which are not mechanically agitated. The dissolved oxygen concentration is measured continuously following a step down or a step up in aeration rate. The response curve is analyzed to obtain the value of K_La Experiments were carried out at several different air flow rates using mixed culture in concurrent tower fermentors with motionless mixers. The effect of sieve trays and Koch motionless mixers on oxygen transfer was investigated using a 3 in. diameter column. The values of K_L aobtained at the bottom of each column were found to be higher than those obtained at the top. Comparison of the results showed that the values ofK_L a were higher when the Koch mixers were used than when the sieve trays were employed. The oxygen uptake rate by the organisms rX, is also calculated by using the K_L a values obtained. They compare favorably withthe experimentally measured values.     

Real-time fluid dynamics investigation and physiological response for erythromycin fermentation scale-up from 50 L to 132 m<Superscript>3</Superscript> fermenter

The physiological response of erythromycin fermentation scale-up from 50 L to 132 m³ scale was investigated. A relatively high oxygen uptake rate (OUR) in early phase of fermentation was beneficial for erythromycin biosynthesis. Correspondingly, the maximal consistency coefficient (K) reflecting non-Newtonian fluid characteristics in 50 L and 132 m³ fermenter also appeared in same phase. Fluid dynamics in different scale bioreactor was further investigated by real-time computational fluid dynamics modeling. The results of simulation showed that the impeller combination in 50 L fermenter could provide more modest flow field environment compared with that in 132 m³ fermenter. The decrease of oxygen transfer rate (OTR) in 132 m³ fermenter was the main cause for impairing cell physiological metabolism and erythromycin biosynthesis. These results were helpful for understanding the relationship between hydrodynamic environment and physiological response of cells in bioreactor during the scale-up of fermentation process.     

Study of the oxygen transfer in a disposable flexible bioreactor with surface aeration in vibrated medium

The oxygen mass transfer is a critical design parameter for most bioreactors. It can be described and analyzed by means of the volumetric mass transfer coefficient K _L a. This coefficient is affected by many factors such as geometrical and operational characteristics of the vessels, type, media composition, rheology and microorganism’s morphology and concentration. In this study, we aim to develop and characterize a new culture system based on the surface aeration of a flexible, single-used bioreactor fixed on a vibrating table. In this context, the K _L a was evaluated using a large domain of operating variables such as vibration frequency of the table, overpressure inside the pouch and viscosity of the liquid. A novel method for K _L a determination based on the equilibrium state between oxygen uptake rate and oxygen transfer rate of the system at given conditions was also developed using resting cells of baker’s fresh yeast with a measured oxygen uptake rate of 21 mg g⁻¹ h⁻¹ (at 30°C). The effect of the vibration frequency on the oxygen transfer performance was studied for frequencies ranging from 15 to 30 Hz, and a maximal K _L a of 80 h⁻¹ was recorded at 30 Hz. A rheological study of the medium added with carboxymethylcellulose at different concentrations and the effect of the liquid viscosity on K _L a were determined. Finally, the mixing time of the system was also measured using the pH method.     

Dynamic measurement of the volumetric oxygen transfer coefficient in fermentation systems

A rapid and internally consistent technique has been developed to measure the volumetric oxygen transfer coefficient, k_La, in fermentation systems. The method consists of tracing the dissolved O₂ concentration of the fermentation broth during a short interruption of the aeration. The O₂ concentration trace thus obtained can be analyzed to determine the values of k_La. Additional experiments on prolonged O₂ starvation, carried out to find the limitation of the technique, suggest that O₂ uptake rate will vary if a prolonged (2–10 min.) O₂ starvation occurs.     

Studies on mass transfer characteristics of a modified airlift fermenter

An approach to modify external loop airlift bioreactor is presented that examines its performance with respect to mass transfer. There are various designs of airlift fermenter [1]. In the proposed system [2] the riser has been replaced by a tube of irregular geometry, in the form of converging-diverging sections (CDT-ALF), so that better mass transfer may be obtained due to better liquid mixing caused by the bubble flow, pulsation effect and early transition to turbulence. Mass transfer characteristics of the modified airlift fermenter CDT-ALF were studied and compared with those of a conventional one, UT-ALF. Overall volumetric mass transfer coefficient,K _L a, was determined by sulfite oxidation method.K _L a was determined with respect toU _G for differenth _i. HigherK _L a was always observed in CDT-ALF compared to that in UT-ALF under any operating condition ofh _i andU _G. If theK _L a values are compared in both the systems under their optimum conditions ofh _i andU _G, CDT-ALF showed 122.5% higher values ofK _L a compared to UT-ALF. However, when both the systems were operated at the lowest experimental conditions ofU _G, thek _L a in CDT-ALF was found to be 170% higher. In UT-ALF while with the decrease ofU _G,k _L a decreased, in CDT-ALF the reverse was observed i.e. at lowU _G,K _L a was higher. However with the increase ofh _i,K _L a decreased in both the systems. To predict volumetric mass transfer coefficientK _L a, empirical correlations were developed by dimensional analysis for both the reactors. The correlations were experimentally verified to determine their reliability to predict mass transfer coefficient and the deviation was found within reasonable limit.List of abbreviations ALF Airlift Fermenter - UT-ALF Uniform Tube Airlift Fermenter - CDT-ALF Converging-diverging Tube Airlift Fermenter     

Investigation of the bacitracin biosynthesis in an airlift bioreactor

Results of pilot plant studies using an external-loop airlift bioreactor (170 l fermentation volume, liquid height-to-riser diameter: 27, loop-to-tower cross-section-area: 0.1225) have proven the relative merits of such a system in the bacitracin biosynthesis produced by the Bacillus licheniformis submerged aerobic cultivation. The results were compared to those obtained in a pilot-scale stirred-tank bioreactor with the same values of k_La. Excepting the aeration rate of 0.2 vvm, the fermentation process performed at 0.5 vvm and 1/0 vvm, respectively, unfolded similarly in the two fermentation devices with respect to the cell mass production, substrate utilization and bacitracin production during the fermentation process. In the riser section of the airlift bioreactor, the dissolved oxygen levels were higher, while in the downcomer section they were lower than those realized in the stirred tank bioreactor. Power requirements of the airlift fermenter were by 17–64% lower than those for a mechanically agitated system, depending on the aeration rates, which led to an important energy saving. Moreover, the lack of mechanical devices in the airlift system provides safety and a more gentle environment for the cultivation of microorganisms.     

Using CFD simulations and statistical analysis to correlate oxygen mass transfer coefficient to both geometrical parameters and operating conditions in a stirred-tank bioreactor

Optimization of a bioreactor design can be an especially challenging process. For instance, testing different bioreactor vessel geometries and different impeller and sparger types, locations, and dimensions can lead to an exceedingly large number of configurations and necessary experiments. Computational fluid dynamics (CFD), therefore, has been widely used to model multiphase flow in stirred-tank bioreactors to minimize the number of optimization experiments. In this study, a multiphase CFD model with population balance equations are used to model gas–liquid mixing, as well as gas bubble distribution, in a 50 L single-use bioreactor vessel. The vessel is the larger chamber in an early prototype of a multichamber bioreactor for mammalian cell culture. The model results are validated with oxygen mass transfer coefficient (k_La) measurements within the prototype. The validated model is projected to predict the effect of using ring or pipe spargers of different sizes and the effect of varying the impeller diameter on k_La. The simulations show that ring spargers result in a superior k_La compared to pipe spargers, with an optimum sparger-to-impeller diameter ratio of 0.8. In addition, larger impellers are shown to improve k_La. A correlation of k_La is presented as a function of both the reactor geometry (i.e., sparger-to-impeller diameter ratio and impeller-to-vessel diameter ratio) and operating conditions (i.e., Reynolds number and gas flow rate). The resulting correlation can be used to predict k_La in a bioreactor and to optimize its design, geometry, and operating conditions.     

Automated fermentation equipment II. Computer–fermentor system

An expensive computer-operated system suitable for data collection and steady-state optimum control of fermentation processes is presented. With this system, minimum generation time has been determined as a function of temperature and pH in the turbidostat cultivation of a yeast strain. The applicability of the computer-fermentor system is also presented by the determination of the dynamic K_L a value.     

Zur Maßstabsübertragung von aeroben mikrobiologischen Prozessen Teil I. Stofftransport

Because the interior scale of turbulence in reinforced bioreactors is essentially greater than the particles of bacteria or yeasts, the mass transfer occurs at the particles only in consequence of the molecular diffusion and depends on the concentration of the soluted material only. As a decided criterion for the transmission of scale in such processes is considered the volumetric oxygen transfer coefficient K_La This article practises a theoretical analysis of the characteristic hydrodynamic conditions in bubble columns and in agitators: the content of gas, the specific area of mass transfer and the coefficient of mass transfer K_L. These conditions are joined with the evaluation of the K_La value. Resultant the data of calculation hence it follows a computational determination of the K_La value by means of the physical matter values, the construction of devises and the process conditions.     

High bioreactor production and emulsifying activity of an unusual exopolymer by Chromohalobacter canadensis 28

Unusual composition of an exopolymer (EP) from an obligate halophilic bacterium Chromohalobacter canadensis 28 has triggered an interest in development of an effective bioreactor process for its production. Its synthesis was investigated in 2‐L bioreactor at agitation speeds at interval 600‐1000 rpm, at a constant air flow rate of 0.5 vvm; aeration rates of 0.5, 1.0, and 1.5 vvm were tested at constant agitation rate of 900 rpm. EP production was affected by both, agitation and aeration. As a result twofold increase of EP yield was observed and additionally increased up to 3.08 mg/mL in a presence of surfactants. For effective scale‐up of bioreactors mass transfer parameters were estimated and lowest values of K_La obtained for the highest productivity fermentation was established. Emulsification activity of EP exceeded that of trade hydrocolloids xanthan, guar gum, and cellulose. A good synergism between EP and commercial cellulose proved its potential exploration as an enhancer of emulsifying properties of trade emulsions. A pronounced lipophilic effect of EP was established toward olive oil and liquid paraffin. Cultivation of human keratinocyte cells (HaCaT) with crude EP and purified γ‐polyglutamic acid (PGA) showed higher viability than control group.     

The volumetric oxygen mass transfer coefficient in antibiotic biosynthesis liquids

This paper approaches the problem of oxygen mass transfer. This transfer is in antibiotic biosynthesis liquids produced by microorganisms belonging to the actinomycete and fungi classes, which exhibit a shear thinning non-Newtonian rheological behaviour. The volumetric oxygen mass transfer coefficients in these liquids (k_L a_b) change during biosynthesis processes. The change is mainly due to rheological parameter modifications, such as increasing the consistency index (K) and decreasing the flow behaviour index (n). The values of k_L a_b were 3.0–6.5 times lower than those recorded in water, and their decreasing depended on the k_L a values obtained without biological liquid and on the nature of fermentation broths, as well. Starting from experimental data, two correlations were established between k_L a_b and P/V,υ_SG and P/V,υ_SG, N, respectively. These correlations contain a dimensionless factor (η_am/η_g), which takes into account the rheological properties of the liquid phase and offers the possibility for a fast and sufficiently accurate estimation of k_L a_b. The empirical correlations developed in the paper correspond reasonably well with the relatively wide variety of experimental data, as in the model proposed by PEREZ and SANDALL , and allow for the comparison of the fermentation batches of the same or different microorganisms; also, they may be applied to the workings of design, scale-up, control and monitoring of bioreactors.     

Optimization of culture conditions for the production of an extracellular ribonuclease by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a benchtop bioreactor

SummarySelf-directing optimization was successfully employed to determine the optimal combination of engineering parameters, viz., pH, aeration rate and agitation rate, for extracellular ribonuclease production by Aspergillus niger SA-13-20 in a batch bioreactor. Maximal RNase production of 5.38 IU ml^–1 was obtained at controlled pH of 2.33, aeration rate of 1.67 v/v/m and agitation rate of 850 rev/min. The effect of oxygen on the fermentation was also investigated. With increase in volumetric oxygen transfer coefficients (K_La), cell growth and RNase production first increased and then decreased. RNase production was further increased to 7.10 IU ml^–1 and the fermentation time was shortened from 96 to 72 h by controlling dissolved oxygen concentration at 10% saturation by aerating oxygen after about 28 h of fermentation under the above optimal condition. The kinetic model showed that RNase production by A. niger SA-13-20 was growth-associated.     

A micro-jet array for economic intensification of gas transfer in bioreactors

Bioreactors are of interest for gas-to-liquid conversion of stranded or waste industrial gases, such as CO, CH₄, or syngas. Process economics requires reduction of bioreactor cost and size while maintaining intense production via rapid delivery of gases to the liquid phase (i.e., high k_La). Here, we show a novel bioreactor design that outperforms all known technology in terms of gas transfer energy efficiency (k_La per power density) while operating at high k_La (i.e., near 0.8 s⁻¹). The reactor design uses a micro-jet array to break feedstock gas into a downward microbubble flow. Hydrodynamic and surfactant measurements show the reactor's advanced performance arises from its bubble breakage mechanism, which limits fluid shear to a thin plane located at an optimal location for bubble breakage. Power dissipation and k_L are shown to scale with micro-jet diameter rather than reactor diameter, and the micro-jet array achieves improved performance compared to classical impinging-jets, ejector, or U-loop reactors. The hydrodynamic mechanism by which the micro-jets break bubbles apart is shown to be shearing the bubbles into filaments then fragmentation by surface tension rather than “cutting in half” of bubbles. Guided by these hydrodynamic insights, strategies for industrial design are given. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2710, 2019