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

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

Growth characteristics of Pleurotus ostreatus in bioreactors

Pleurotus ostreatus was cultured in a bioreactor equipped with different impeller geometries under non-limiting nutrient conditions. With a Rushton turbine impeller the specific growth rate decreased 30% and pellet diameter was reduced 15% when the aeration rate was increased from 1 to 1.5 vvm. Agitation rate reduced the pellet diameter from 5.1 mm to 2.8 mm using 200 rpm and 400 rpm of agitation, respectively. Specific growth rates of 0.036, 0.020 and 0.041 h–1 were obtained with Roshton (disc turbine), Helical Ribbon and InterMIG impellers, respectively. Impeller geometry is important to control the pellet size and consequently growth rate of P. ostreatus.     

Biological responses of hybridoma cells to hydrodynamic shear in an agitated bioreactor.

Effects of long-term hydrodynamic shear on hybridoma cells were investigated in a 250-ml continuous stirred-tank reactor (CSTR). Cells grown at steady state were subjected to step changes in agitation rates. Cell viability, glucose consumption, and monoclonal antibody (MAb) production were determined at high agitation rates and compared with the control (100 rev min-1). Impeller tip speeds higher than 40 cm s-1 caused a significant drop in cell concentration and respiration activity, and increased lactate dehydrogenase (LDH) release to the culture medium. Also, high agitation speeds caused a decrease in MAb concentration and an increase in specific glucose consumption rate. The effects of dilution rate and serum concentration on the sensitivity of hybridoma cells to hydrodynamic shear were determined. Serum was found to protect the cells against shear damage and had a significant positive effect on hybridoma growth and MAb production. Shear damage on cells in CSTR was approximated to first-order kinetics. The death rate constant increased sharply at impeller tip speeds above 40 cm s-1.     

Mixing state of xanthan gum solution in an aerated and agitated fermentor—Effects of impeller size on volumes of mixing regions and circulation time distribution

The effects of the impeller diameter and width on the volumes of the micromixing and macromixing regions, and on the circulation time distribution were investigated at various agitation speeds to formulate the relationships of them in emperical equations. A fermentor was a 10-l capacity, which was equipped with a turbine impeller with six flat balades and aerated at 1 vvm. It was found that the volumes of the micromixing and macromixing regions depended on the tip speed of the impeller, ND, and the discharging performance of the impeller, ND²W, respectively, in the xabthan gum solution with concentrations of 0.9, 1.8, 2.7, and 3.9%. Empirical equations were derived to estimate the volume of each mixing region from the impeller diameter, D, impeller width, W, agitation speed, N, and consistency coefficient of the xanthan gum solution. On the other hand, the circulation time distribution could be estimated empirically from only the impeller diameter and agitation speed, regardless of variation in the impeller width and consistency coefficient of the xanthan gum solution tested.     

Dependence of penicillium chrysogenum growth, morphology, vacuolation, and productivity in fed-batch fermentations on impeller type and agitation intensity

The influence of the agitation conditions on the growth, morphology, vacuolation, and productivity of Penicillium chrysogenum has been examined in 6 L fed-batch fermentations. A standard Rushton turbine, a four-bladed paddle, and a six-bladed pitched blade impeller were compared. Power inputs per unit volume of liquid, P/VL, ranged from 0.35 to 7.4 kW/m3. The same fermentation protocol was used in each fermentation, including holding the dissolved oxygen concentration above 40% air saturation by gas blending. The mean projected area (for all dispersed types, including clumps) and the clump roughness were used to characterize the morphology. Consideration of clumps was vital as these were the predominant morphological form. For a given impeller, the batch-phase specific growth rates and the overall biomass concentrations increased with agitation intensity. Higher fragmentation at higher speeds was assumed to have promoted growth through increased formation of new growing tips. The mean projected area increased during the rapid growth phase followed by a sharp decrease to a relatively constant value dependent on the agitation conditions. The higher the speed, the lower the projected area for a given impeller type. The proportion by volume of hyphal vacuoles and empty regions decreased with speed, possibly due to fragmentation in the vacuolated regions. The specific penicillin production rate was generally higher with lower impeller speed for a given impeller type. The highest value of penicillin production as well as its rate was obtained using the Rushton turbine impeller at the lowest speed. At given P/VL, changes in morphology, specific growth rate, and specific penicillin production rate depended on impeller geometry. The morphological data could be correlated with either tip speed or the "energy dissipation/circulation function," but a reasonable correlation of the specific growth rate and specific production rate was only possible with the latter. Copyright 1998 John Wiley & Sons, Inc.     

Enhancement of glucose oxidase production in batch cultivation of recombinant Saccharomyces cerevisiae: optimization of oxygen transfer condition

AIMS: To obtain an optimal combination of agitation speed and aeration rate for maximization of specific glucose oxidase (GOD) production in recombinant Saccharomyces cerevisiae, and to establish a correlation between kLa vis-à-vis oxygen transfer condition and specific glucose oxidase production. METHODS AND RESULTS: The oxygen transfer condition was manifested indirectly by manipulating the impeller speed and aeration rate in accordance with a Central Composite Rotatory Design (CCRD). The dissolved oxygen concentration and the volumetric oxygen transfer coefficient (kLa) were determined at corresponding combinations of impeller speed and aeration rate. The maximal specific extracellular glucose oxidase production (3.17 U mg-1 dry cell mass) was achieved when the initial dissolved oxygen concentration was 6.83 mg l-1 at the impeller speed of 420 rev min-1 and at the rate of aeration of 0.25 vvm. It was found out that while impeller speed had a direct effect on the production of enzyme, a correlation between kLa and specific GOD production could not be established. CONCLUSION: At the agitation speed of 420 rev min-1 and at 0.25 vvm aeration rate, the degree of turbulence and the dissolved oxygen concentration were thought to be optimal both for cellular growth and production of enzyme. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined effect of agitation and aeration on recombinant glucose oxidase production in batch cultivation has not yet been reported in the literature. Therefore, this study gives an insight into the effect of these two important physical parameters on recombinant protein production. It also suggests that since there is no correlation between kLa and specific production of GOD, kLa should not be used as one of the scale-up parameters.     

The role of intercellular contacts in the initiation of growth and in the development of a transiently nonculturable state by the cultures of Rhodococcus rhodochrous grown in poor media

It was found that the growth of Rhodococcus rhodochrous cells in modified Saton's medium strongly depends on the rate of culture agitation in the flask: an agitation at 250 rpm in flasks with baffles stops cell multiplication, whereas slight agitation leads to pronounced culture growth. The growth retardation phenomenon was reversible and did not manifest itself in exponential-phase cultures or when the cells were grown in a rich medium; furthermore, it was not connected with the degree of culture aeration. When agitated at a moderate rate, the bacterial cells formed aggregates in the lag phase, which broke up into single cells in the exponential phase. The inhibitory effect of vigorous agitation was removed by the addition to the medium of the supernatant (SN) of a log-phase culture grown in the same medium with moderate agitation. Vigorous agitation is thought to interfere with the cell contacts, whose establishment is necessary for the development of an R. rhodochrous culture in a poor medium, which occurs in the form of (micro) cryptic growth. When grown in modified Saton's medium, R. rhodochrous cells were capable of transition, in the prolonged stationary phase, to a resting and transiently nonculturable state. Such cells could be resuscitated by incubation in a liquid medium with the addition of the supernatant or the Rpf secreted protein. The formation of transiently nonculturable cells was only possible under the conditions of a considerable agitation rate (250-300 rpm), which prevented secondary (cryptic) growth of the culture. This circumstance indicates the importance of intercellular contacts not only for the initiation of growth but also for the transition of the bacteria to a dormant state.     

Mechanistic analysis of xanthan gum production in a stirred tank

The overall effect of agitation on xanthan gum production by Xanthomonas campestris ATCC13951 in a stirred vessel was mechanistically analyzed considering local variation of the specific production rate due to variation of shear stress in the vessel. The whole liquid volume in a fermentor was roughly divided into three regions; the micromixing region around the impeller with high shear stress, the macromixing region dominated by a circulating flow and the stagnant region. The value of the shear rate was first ascertained by experiments in order to obtain a picture of shear rate variation in a radial direction from the impeller, and the equivalence between the volumes of the high shear stress region and micromixing region was confirmed. The shear stress obtained using a correlation between the shear rate at the impeller tip and Reynolds number of Wichterle et al. was used as a representative of the shear stress in the micromixing region, and the shear stress estimated by use of an empirical correlation between the average shear rate in a fermentor and agitation speed derived by Metzner et al. was adopted as a representative of the shear stress in the macromixing region. The information about the circulation time distribution was also used to take into account oxygen deficiency during circulation of liquid elements in the macromixing region, considering that oxygen from the gas phase was supplied mainly in the high shear region. The calculated values of xanthan gum concentrations which were obtained by the proposed simulation method agreed well with the experimental data in the time course of xanthan gum production at various agitation speeds. Experimental results of the relationship between the overall specific production rate and ND (N, agitation speed, and D, impeller diameter) was also verified by the proposed method.     

Study of hydrodynamics in microcarrier culture spinner vessels: A particle tracking velocimetry approach

Three-dimensional particle tracking velocimetry (3-D PTV), a modern, quantitative, visualization tool, has been applied to the characterization of the flow field in the impeller region of cell culture reactor vessels. The experimental system used here is a 250-mL microcarrier spinner vessel. The studies were conducted at three different agitation rates, 90, 150, and 210 rpm, corresponding to healthy, mildly damaging, and severely damaging shear intensities, respectively. The flow can be classified into three regions: a predominantly tangential (azimuthal) flow generated by the impeller; a trailing vortex region coming off the impeller tip; and a converging flow region close to the center of the vessel. The latter two are the regions of highest velocity gradients. Energy dissipation rates due to mean velocity gradients were also calculated to characterize the impeller stream. Local specific energy dissipation rates > 10,000 erg/(cm(3)sec) . have been measured. It is proposed that the critical regions for microcarrier culture damage due to impeller hydrodynamics are the trailing vortex region and the high energy converging flow region. Graphical representation of the mean velocity flow fields and the distribution of energy dissipation rates in the impeller region are also presented here. The merits of using the dissipation function (measure of specific energy dissipation rate) as a possible scale-up parameter are also discussed. (c) 1996 John Wiley & Sons, Inc.     

Effect of agitation rate and impeller design on oxygen transfer coefficients in small bioreactors using surface aeration

The effects of agitation rate and impeller type on the combined oxygen mass-transfer coefficient (kL a) in four different benchtop bioreactors have been examined. Surface oxygenation of a cell culture medium supplemented with fetal bovine serum and distilled deionized water has been studied by passing air through the bioreactor headspace at approximately one headspace volume per minute. A new ribbon-type impeller design using strips of Teflon has been shown to be superior to conventional impeller designs for oxygen transfer.     

Effect of dissolved oxygen concentration and impeller tip speed on itaconic acid production by Aspergillus terreus

Summary Effect of aeration rate and impeller tip speed on mycelium growth and itaconic acid production was investigated in a batch culture of Aspergillus terreus IFO-6365. When impeller tip speed was 94.2 cm/sec at a fixed aeration rate of 0.5 vvm, itaconic acid concentration was 3.6 and 1.6 times higher than those in the impeller tip speed of 62.8 and 125.7 cm/sec, respectively. When an oxygen-enriched air was supplied at a fixed impeller tip speed of 94.2 cm/sec and dissolved oxygen concentration was maintained in the 20–60 % range, both itaconic acid concentration and mycelium growth were not affected by the dissolved oxygen concentration.     

采用计算流体力学技术研究搅拌对兽疫链球菌发酵生产透明质酸的影响

搅拌是影响透明质酸(HA)发酵的一个重要因素,然而有关搅拌对HA发酵影响的认识存在较大争议。本研究采用计算流体力学(CFD)技术深入研究了搅拌对菌体生长和HA合成的影响。结果表明,菌体量和HA产量受搅拌转速的影响很小,而HA分子量随着转速的增加呈现出先增加后降低的趋势。分阶段控制转速研究表明转速对HA分子量的影响主要体现在HA合成阶段。CFD计算结果表明随着搅拌转速的增加,混合时间降低的同时反应器内部的剪切速率明显增加。最终通过改变搅拌桨组合方式的手段有效地解决了上述矛盾,并使得HA分子量提高23.9%。     

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

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

Effects of agitation intensity on mycelial morphology and protein production in chemostat cultures of recombinant Aspergillus oryzae

The effects of agitation on fragmentation of a recombinant strain of Aspergillus oryzae and its consequential effects on protein production have been investigated. Constant mass, 5.3-L chemostat cultures at a dilution rate of 0.05 h-1 and a dissolved oxygen level of 75% air saturation, have been conducted at 550, 700, and 1000 rpm. These agitation speeds were chosen to cover a range of specific power inputs (2.2 to 12 kW m-3) from realistic industrial levels to much higher values. The use of a constant mass chemostat linked to a gas blender allowed variation of agitation speed and hence gas hold-up without affecting the dilution rate or the concentration of dissolved oxygen. The morphology of both the freely dispersed mycelia and clumps was characterized using image analysis. Statistical analysis showed that it was possible to obtain steady states with respect to morphology. The mean projected area at each steady state under growing conditions correlated well with the 'energy dissipation/circulation" function, [P/(kD3tc)], where P is the power input, D the impeller diameter, tc the mean circulation time, and k is a geometric constant for a given impeller. Rapid transients of morphological parameters in response to a speed change from 1000 to 550 rpm probably resulted from aggregation. Protein production (alpha-amylase and amyloglucosidase) was found to be independent of agitation speed in the range 550 to 1000 rpm (P/V = 2.2 and 12.6 kW m-3, respectively), although significant changes in mycelial morphology could be measured for similar changes in agitation conditions. This suggests that mycelial morphology does not directly affect protein production (at a constant dilution rate and, therefore, specific growth rate). An understanding of how agitation affects mycelial morphology and productivity would be valuable in optimizing the design and operation of large-scale fungal fermentations for the production of recombinant proteins. Copyright 1999 John Wiley & Sons, Inc.     

Damaging agitation intensities increase DNA synthesis rate and alter cell-cycle phase distributions of CHO cells

The effects of fluid-mechanical force (agitation) on the cell cycle kinetics of Chinese hamster ovary (CHO) cells cultured in suspension in 2-L bioreactors has been examined. A two-color flow cytometry method was used to determine the fraction rate of DNA synthesis. With increased agitation intensity, cell viability decreased as a result of increased cell death. However, increased agitation induced the viable cells of the culture to a higher proliferative state relative to a control culture. The fraction of viable cells of the high-agitation culture (250 rpm) in S phase was higher (up to 45%) and in G1 phase was lower (up to 50%) compared with the viable cells of the control culture (80 rpm). The DNA synthesis rate per viable S-phase cell of the high-agitation culture was confirmed by recovery experiments, which were conducted to measure the apparent specific growth rate and the cell cycle kinetics of the high-agitation culture upon reduction in the agitation rate from 250 rpm back to 80 rpm. The apparent specific growth rate of the test culture, calculated for the first 12 h of the recovery period, was greater than the apparent specific growth rate of the control culture. Furthermore, the proliferative state of the viable cells of the test culture, which had become higher relative to the control culture during the high agitation period, gradually approached the level of the control culture during recovery. Results also show that the magnitude of the agitation intensity; the culture agitated at 250 rpm attained a greater proliferative state than a parallel culture agitated at 235 rpm. The 250-rpm culture had a higher fraction of S-phase and a lower fraction of G1-phase cells than the 235-rpm culture. The DNA sunthesis rate per viable S-phase cell of the 250-rpm culture was greater than of the 235-rpm culture. (c) 1992 John Wiley & Sons, Inc.     

Pellet morphology, culture rheology and lovastatin production in cultures of Aspergillus terreus

Pellet growth of Aspergillus terreus ATCC 20542 in submerged batch fermentations in stirred bioreactors was used to examine the effects of agitation (impeller tip speed u(t) of 1.01-2.71 ms(-1)) and aeration regimens (air or an oxygen-enriched mixture containing 80% oxygen and 20% nitrogen by volume) on the fungal pellet morphology, broth rheology and lovastatin production. The agitation speed and aeration methods used did not affect the biomass production profiles, but significantly influenced pellet morphology, broth rheology and the lovastatin titers. Pellets of approximately 1200 microm initial diameter were reduced to a final stable size of approximately 900 microm when the agitation intensity was >/=600 rpm (u(t)>/=2.03 ms(-1)). A stable pellet diameter of approximately 2500 microm could be attained in less intensely agitated cultures. These large fluffy pellets produced high lovastatin titers when aerated with oxygen-enriched gas but not with air. Much smaller pellets obtained under highly agitated conditions did not attain high lovastatin productivity even in an oxygen-enriched atmosphere. This suggests that both an upper limit on agitation intensity and a high level of dissolved oxygen are essential for attaining high titers of lovastatin. Pellet size in the bioreactor correlated equally well with the specific energy dissipation rate and the energy dissipation circulation function. The latter took into account the frequency of passage of the pellets through the high shear regions of the impellers. Pellets that gave high lovastatin titers produced highly shear thinning cultivation broths.     

The Role of Intercellular Contacts in the Initiation of Growth and in the Development of a Transiently Nonculturable State by Cultures of Rhodococcus rhodochrous Grown in Poor Media

It was found that the growth of Rhodococcus rhodochrous cells in a modified Saton’s medium strongly depends on the rate of culture agitation in the flask: agitation at 250 rpm in flasks with baffles stops cell multiplication, whereas slight agitation leads to pronounced culture growth. The growth retardation phenomenon was reversible and did not manifest itself in exponential-phase cultures or when the cells were grown in a rich medium; furthermore, it was not connected with the degree of culture aeration. When agitated at a moderate rate, the bacterial cells formed aggregates in the lag phase, which broke up into single cells in the exponential phase. The inhibitory effect of vigorous agitation was removed by the addition, to the medium, of the supernatant (SN) of a log-phase culture grown in the same medium with moderate agitation. Vigorous agitation is thought to interfere with cell contact, whose establishment is necessary for the development of an R. rhodochrous culture in a poor medium, which occurs in the form of (micro) cryptic growth. When grown in a modified Saton’s medium, R. rhodochrous cells were capable of transition, in the prolonged stationary phase, to a resting and transiently nonculturable state. Such cells could be resuscitated by incubation in a liquid medium with the addition of the supernatant or the Rpf secreted protein. The formation of transiently nonculturable cells was only possible under the conditions of a considerable agitation rate (250–300 rpm), which prevented secondary (cryptic) growth of the culture. This circumstance indicates the importance of intercellular contacts not only for the initiation of growth but also for the transition of the bacteria to a dormant state.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 489–797.Original Russian Text Copyright © 2005 by Voloshin, Shleeva, Syroeshkin, Kaprelyants.     

机械搅拌式动物细胞反应器不同桨型组合的CFD数值模拟与优化

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

Comparison of biotin production by recombinant Sphingomonas sp. under various agitation conditions

Biotin production by fermentation of recombinant Sphingomonas sp./pSP304 was investigated. A complex medium containing 60g/l of glycerol and 30g/l of yeast extract was suitable for biotin production. Biotin was produced in the late logarithmic or stationary phase after glycerol starvation. The optimum pH value for biotin production was 7.0. When the dissolved oxygen concentration (DO) was controlled at a constant level, the biotin concentration produced after 120h was significantly lower than that obtained in a test tube culture. Therefore, a batchwise jar-fermentor culture with a constant agitation speed and without DO control was conducted for investigating the effect of agitation conditions on biotin production. Six types of impeller were tested: turbine-blade type, turbo-lift type, rotating mesh type (EGSTAR((R))), screw with draft tube type, Maxblend((R))type, and anchor type. With some impellers, agitation speed was also changed. Both the maximum cell concentration and biotin production varied depending on agitation conditions. Relatively high cell concentrations were attained with four of the impeller types, turbine-blade type, rotating mesh type, Maxblend((R)) type, and anchor type. Among these impellers, the turbine-blade impeller with sintered sparger was suitable for biotin production. After 120h, the cell concentration reached an OD(660) of 43 and a biotin concentration of 66mg/l was obtained, which was comparable with the results from the test tube culture. Morphological variation was also observed depending on the agitation conditions: oval-shaped, rod-shaped, and elongated-shaped cells. Biotin production was relatively high in slightly long rod-shape cells but low in elongated cells. The difference in morphology appeared to depend on the shear stress. It was found that biotin production was strongly correlated with cell length and the oxygen transfer coefficient (k(L)a); cell lengths in the range 4-7μm and k(L)a values in the range 1.5-2.0/min were found to be suitable for biotin production in jar-fermentor culture.     

Agitation induced cell injury in microcarrier cultures. Protective effect of viscosity is agitation intensity dependent: Experiments and modeling

The effect of medium viscosity on the specific death rate of bovine embryonic kidney (BEK) cells cultured in spinner flask microcarrier cultures has been examined for various impeller speeds. Two types of media were used, a serum-containing growth medium and a serum-free maintenance medium. The latter does not support cell growth. We found that increasing medium viscosity suppresses cell death rates in both growth and maintenance medium cultures in an agitation-intensity-dependent fashion; the beneficial effect of medium viscosity in reducing the specific death rate is amplified as the agitation rate is increased. Furthermore, increasing medium viscosity has no effect on the specific death rate of the cells when the agitation rate is below a critical level. A model based on the turbulent energy content of eddies in the dissipation spectrum of turbulence of length scales on the order of magnitude of the microcarrier diameter and lower has been developed to account for cell death due to both bead-to-bead and bead-to-eddy interactions. The model constitutes a significant departure from previous efforts first because both types of interactions are accounted for simultaneously and second because the properties of a spectrum of eddies instead of the Kolmogorov-scale eddy size alone are used in the model. The model explains the functional dependence of the specific death rates on the medium viscosity at varying agitation intensities.