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.     

Performance of mammalian cell culture bioreactor with a new impeller design

To improve the oxygen transfer in a mammalian cell bioreactor, a new type of impeller consisting of a double-screen concentric cylindrical cage impeller (annular cage impeller in short) was designed and its mass transfer rate evaluated. This new impeller design increases the specific screen area, and the convective mass transfer rate through the annular cage was significantly increased. The oxygen transfer rates with the new impeller and the commercially available cell-lift impeller (CelliGen by New Brunswick Scientific Co.) were evaluated and their performance compared at various rates of aeration and agitation. The results showed that with the new impeller, the oxygen transfer rate was increased by 19% in water and 21% in cell-free culture medium supplemented with 10% horse serum, the total hybridoma cell concentration was increased to 3.4 x 10(7) cells/mL, and the IgG(1) subtype monoclonal antibody (MAb) product concentration was also increased to 512 mg/L in perfusion culture of murine hybridoma cell line 62'D3. These improvements in oxygen transfer rate, cell concentration, and MAb product concentration are all very significant. The mass transfer resistance in the cell-lift impeller system was found to be mainly due to the surface area of the single-screen cage impeller. The new annular cage impeller not only provided the increased surface area for convective oxygen transfer but also protected cells from hydrodynamic shear damage, thereby achieving a significant bioprocess improvement in terms of higher viable cell concentration, higher product concentration, and higher oxygen transfer rate in the mammalian cell bioreactor system.     

Effects of increased impeller power in a production-scale Aspergillus oryzae fermentation

The goal in this study was to determine how increased impeller power affects enzyme expression in large-scale (80 m(3)), fed-batch Aspergillus oryzae fermentations. An approximate 50% increase in average impeller power was achieved by increasing impeller diameter approximately 10%, while operating at slightly reduced speed. Measured decreases in terminal (95%) mixing time show increased power improved bulk mixing. However, batches operated at increased power had lower recombinant enzyme productivity. Biomass assays and image analysis tests showed no significant difference between "high power" and control batches, suggesting that slower growth, altered morphology, or increased hyphal fragmentation were not the cause of reduced productivity. Off-line tests on the shear-thinning, highly viscous broth show oxygen limitation occurred after transport through the air-liquid interface and imply the limitation may involve bulk mixing. Specifically, oxygen transfer may be limited to a small zone surrounding each impeller. When this is the case, oxygen mass transfer will be determined by both impeller shear and fluid circulation, which have been characterized with the energy dissipation/circulation function (EDCF). EDCF values during control fermentations were approximately constant at 25 kW m (-3) s(-1), while EDCF values during "high power" batches fell linearly from 40 to 15 kW m (-3) s(-1). The point at which "high power" EDCF values drop below those in control fermentations corresponds almost exactly with the point at which product titer stops increasing. Thus, our findings suggest oxygen mass transfer was less efficient during the latter half of "high power" fermentations because of reductions in impeller speed and subsequent decreases in EDCF values. This observation has clear implications during the scale-up of viscous fungal fermentations, implying that not only is the level of impeller power important, but also relevant is how this power is applied.     

Proof of concept: predicting the onset of meat spoilage by an integrated oxygen sensor spot in MAP packages

During storage of modified atmosphere packaged (MAP) meat, the initial microbiota grows to high cell numbers, resulting in perceptible spoilage after exceeding a specific threshold level. This study analyses, whether elevated oxygen consumption in the headspace of MA-packages would enable a prediction method for meat spoilage. We monitored the growth of single spoiling species inoculated on high-oxygen MAP beef and poultry, performed sensorial analysis and determined oxygen concentrations of the headspace via a non-invasive sensor spot technology. We detected microbial headspace oxygen consumption occurring prior to perceptible meat spoilage for certain species inoculated on beef steaks. However, headspace oxygen consumption and cell counts at the onset of spoilage were highly species-dependent, which resulted in a strong (Brochothrix thermosphacta) and moderate (Leuconostoc gelidum subspecies) decrease of the headspace oxygen content. No linear decrease of the headspace oxygen could be observed for Carnobacterium divergens and Carnobacterium maltaromaticum inoculated on poultry meat. We demonstrate the applicability of an incorporated oxygen sensor spot technology in MAP meat packages for detection of spoilage in individual packages prior to its perceptible onset. This enables individual package evaluation and sorting within retail, and consequently reduces meat disposal as waste.     

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.     

Mass transfer and mixing rates in fermentation vessels

The effects on mass-transfer and overall mixing rates of varying impeller geometry and operating speed have been studied for flat-bladed turbines in laboratory fermentors, in aerated aqueous solutions, and in unaerated and aerated suspensions (1.6% w/v) of paper pulp. In the absence of suspended solid, oxygen absorption rates could be correlated directly with power input. In the pulp suspension, oxygen absorption at a given power input was influenced by impeller geometry and operating speed. The data for the three-phase system can be correlated by a dimensionless equation relating oxygen-transfer rates and mixing times to the geometrical and operating parameters of the impellers.     

Gas-liquid dispersion with dual Rushton turbine impellers

Aerated and unaerated power consumption and flow patterns in a 0.56 m diameter agitated vessel containing water with dual Rushton turbines have been studied. Under unaerated conditions with a liquid height-to-diameter ratio of 2, an impeller spacing of 2 to 3 times the impeller is required for each to draw an amount of power equal to a single impeller. For aerated conditions, if a similar spacing is used, equations for the flooding-loading transition and for power consumption for a single Rushton impeller can be extended relatively easily to dual systems. All results for this spacing are explained by reference to bulk flow patterns and gassed-filled cavity structures and the proportion of sparged gas flowing through the upper impeller is also estimated. Such a spacing is generally recommended since it maximizes the power draw and hence the potential for oxygen mass transfer. Data are presented for other spacings but the results do not fit in easily with single agitator studies because strong impeller-impeller flow pattern interactions occur.     

On-line gas analysis in animal cell cultivation: I. Control of dissolved oxygen and pH

To monitor gas reaction rates in animal cell culture at constant dissolved oxygen concentration (DO) and constant pH it was necessary to develop improved control methods. Decoupling of both controllrs was obtained by manipulation of molar fractions of oxygen and carbon dioxide in the gas phase. Two pairs of DO and pH controllers were designed and tested both in simulation and exprimental runs. The first controller pair was developed for headspace aeration only, whereas the second controller pair was designed for bubble aeration using a microsparger and flushing the headspace with helium. pH was controlled by a conventional discrete PID controller in its velocity form. For DO control two linear state space feedback controllers with parameter adaptation were established. In these controllers the oxygen uptake rate (OUR) was considered as a disturbance and was not included in the mathematical model. The feedback gain adaptation was based on the difference between the actual molar fraction of oxygen at time step n and the initial molar fraction. This difference is related to OUR and was used to increase or decrease the state feedback controller gain (k and k(1), respectively) in a slow manner. With these controllers it was possible to get an excellent online estimate of OUR. In the case of bubble aeration a simple gas phase mass balance was sufficient, whereas during the headspace aeration a liquid phase balance was required. It has been shown that determination of OUR using gas balance requires a significantly better controller performance compared to just keeping DO and pH within reasonable limits. (c) 1995 John Wiley & Sons, Inc.     

A novel impeller configuration to improve fungal physiology performance and energy conservation for cephalosporin C production

Effects of impeller configuration on fungal physiology and cephalosporin C production were investigated by an industrial strain Acremonium chrysogenum in a 12m(3) bioreactor equipped with conventional and novel impeller configuration, respectively. The cell growth and oxygen uptake rate (OUR) profiles were little affected by the impeller configurations. However, differing impeller combinations significantly affected the morphology, which in turn influenced cephalosporin C production. Under the novel impeller configuration, the production of cephalosporin C was 10% higher and an excessive amount of dispersed arthrospores was also observed. Computational fluid dynamics (CFD) simulation further revealed that poor mass and energy exchange as well as inhomogeneous environment existed in the bioreactor equipped with conventional impeller configuration. For equivalent power dissipation, the volume oxygen transfer coefficient (K(L)a) could be enhanced by 15% compared with that of conventional impeller configuration. Power consumption was dramatically decreased by 25% by using novel impeller configuration.     

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.     

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.     

Sparging and agitation-induced injury of cultured animals cells: Do cell-to-bubble interactions in the bulk liquid injure cells?

It has been established that the forces resulting from bubbles rupturing at the free air (gas)/liquid surface injure animal cells in agitated and/or sparged bioreactors. Although it has been suggested that bubble coalescence and breakup within agitated and sparged bioreactors (i.e., away from the free liquid surface) can be a source of cell injury as well, the evidence has been indirect. We have carried out experiments to examine this issue. The free air/liquid surface in a sparged and agitated bioractor was eliminated by completely filling the 2-L reactor and allowing sparged bubbles to escape through an outlet tube. Two identical bioreactors were run in parallel to make comparisons between cultures that were oxygenated via direct air sparging and the control culture in which silicone tubing was used for bubble-free oxygenation. Thus, cell damage from cell-to-bubble interactions due to processes (bubble coalescence and breakup) occurring in the bulk liquid could be isolated by eliminating damage due to bubbles rupturing at the free air/liquid surface of the bioreactor. We found that Chinese hamster ovary (CHO) cells grown in medium that does not contain shear-protecting additives can be agitated at rates up to 600 rpm without being damaged extensively by cell-to bubble interactions in the bulk of the bioreactor. We verified this using both batch and high-density perfusion cultures. We tested two impeller designs (pitched blade and Rushton) and found them not to affect cell damage under similar operational conditions. Sparger location (above vs. below the impeller) had no effect on cell damage at higher agitation rates but may affect the injury process at lower agitation intensities (here, below 250 rpm). In the absence of a headspace, we found less cell damage at higher agitation intensities (400 and 600 rpm), and we suggest that this nonintuitive finding derives from the important effect of bubble size and foam stability on the cell damage process. (c) 1996 John Wiley & Sons, Inc.     

Development, parallelization, and automation of a gas-inducing milliliter-scale bioreactor for high-throughput bioprocess design (HTBD)

A novel milliliter-scale bioreactor equipped with a gas-inducing impeller was developed with oxygen transfer coefficients as high as in laboratory and industrial stirred-tank bioreactors. The bioreactor reaches oxygen transfer coefficients of >0.4 s(-1). Oxygen transfer coefficients of >0.2 s(-1) can be maintained over a range of 8- to 12-mL reaction volume. A reaction block with integrated heat exchangers was developed for 48-mL-scale bioreactors. The block can be closed with a single gas cover spreading sterile process gas from a central inlet into the headspace of all bioreactors. The gas cover simultaneously acts as a sterile barrier, making the reaction block a stand-alone device that represents an alternative to 48 parallel-operated shake flasks on a much smaller footprint. Process control software was developed to control a liquid-handling system for automated sampling, titration of pH, substrate feeding, and a microtiter plate reader for automated atline pH and atline optical density analytics. The liquid-handling parameters for titration agent, feeding solution, and cell samples were optimized to increase data quality. A simple proportional pH-control algorithm and intermittent titration of pH enabled Escherichia coli growth to a dry cell weight of 20.5 g L(-1) in fed-batch cultivation with air aeration. Growth of E. coli at the milliliter scale (10 mL) was shown to be equivalent to laboratory scale (3 L) with regard to growth rate, mu, and biomass yield, Y(XS).     

Mass transfer and hydrodynamic characteristics of unbaffled stirred bio-reactors: Influence of impeller design

Unbaffled stirred tanks are increasingly recognized as a viable alternative to common baffled tanks for a range of processes where the presence of baffles is undesirable for some reason. For instance, in the case of shear sensitive cell cultivation (e.g. human cells), unbaffled tanks have been recently found to be able to provide sufficient mass transfer through the free surface vortex. As a consequence the need for bubble formation and subsequent bursting, along with relevant cells damage, is conveniently avoided. In this work the influence of impeller geometry on mass transfer performance and power demand of an unbaffled stirred vessel operating both in sub-critical conditions (the free surface vortex has not yet reached the impeller) and in super-critical conditions (the free surface vortex has reached the impeller and a gas phase is ingested and dispersed inside the reactor) is presented.Experimental results show that the mass transfer performance of unbaffled systems is mainly affected by specific power consumption. Among the stirrer geometries investigated a simple PBT was found to provide the most interesting oxygen transfer performance in the sub-critical regime, and can therefore be regarded as a particularly suitable stirrer for shear sensitive cultures. As regards the super-critical regime, unbaffled tanks are found to provide a performance comparable with that of the standard (baffled) bioreactors, hence resulting in a viable alternative also for fermentations involving robust cells.     

Oxygen transfer and energy dissipation rate in surface aerator

The dissipation rate of turbulent kinetic energy (ε) is a key parameter for mixing in surface aerators. In particular, determination ε across the impeller stream, where the most intensive mixing takes place, is essential to ascertain that an appropriate degree of mixing is achieved. Present work by using commercial software VisiMix^® calculates the energy dissipation rate in geometrically similar unbaffled surface aeration systems in order to scale-up the oxygen transfer process. It is found that in geometrically similar system, oxygen transfer rate is uniquely correlated with dissipation rate of energy. Simulation or scale-up equation governing oxygen transfer rate and dissipation rate of energy has been developed in the present work.     

翼形轴流桨在红霉素发酵中的应用研究

在20t工业发酵罐中,研究了翼形轴流桨搅拌对红霉素发酵过程的影响。重点考察了粘度,溶氧,效价等过程参数变化,以及搅拌功耗与发酵产量之间的关系。研究结果表明：（1）与传统的涡轮搅拌桨相比,翼形轴流桨搅拌其发酵过程参数（粘度,溶氧,效价等）随时间的变化曲线有明显的差异;（2）在相同的生产条件下,用翼形轴流桨代表涡轮桨可有效提高生产效率。     

A comprehensive comparison of mixing, mass transfer, Chinese hamster ovary cell growth, and antibody production using Rushton turbine and marine impellers

Large scale production of monoclonal antibodies has been accomplished using bioreactors with different length to diameter ratios, and diverse impeller and sparger designs. The differences in these physical attributes often result in dissimilar mass transfer, mechanical stresses due to turbulence and mixing inside the bioreactor that may lead to disparities in cell growth and antibody production. A rational analysis of impeller design parameters on cell growth, protein expression levels and subsequent antibody production is needed to understand such differences. The purpose of this study was to examine the impact of Rushton turbine and marine impeller designs on Chinese hamster ovary (CHO) cell growth and metabolism, and antibody production and quality. Experiments to evaluate mass transfer and mixing characteristics were conducted to determine if the nutrient requirements of the culture would be met. The analysis of mixing times indicated significant differences between marine and Rushton turbine impellers at the same power input per unit volume of liquid (P/V). However, no significant differences were observed between the two impellers at constant P/V with respect to oxygen and carbon dioxide mass transfer properties. Experiments were conducted with CHO cells to determine the impact of different flow patterns arising from the use of different impellers on cell growth, metabolism and antibody production. The analysis of cell culture data did not indicate any significant differences in any of the measured or calculated variables between marine and Rushton turbine impellers. More importantly, this study was able to demonstrate that the quality of the antibody was not altered with a change in the impeller geometry.     

Application of Oxygen-Enriched Aeration in the Conversion of Glycerol to Dihydroxyacetone by Gluconobacter melanogenus IFO 3293

Gluconobacter melanogenus 3293 converts glycerol to dihydroxyacetone(DHA) during exponential growth on a yeast extract-phosphate medium at pH 7. The efficiency of this conversion in 25-liter batch fermentations has been found to increase over threefold, when oxygen tension is controlled by increasing the partial pressure of oxygen in the aeration. Conversion of glycerol to DHA does not occur under oxygen-limited fermentation conditions. When the dissolved oxygen tension was maintained at 0.05 atmospheres (using oxygen-enriched air), quantitative conversion of up to 100 g of glycerol/liter to DHA was obtained in 33 h. The amount of glycerol converted can be increased without increasing impeller speed or aeration rate. This increase is not the result of increased production of cell mass. The specific conversion of glycerol to DHA increased from 12.2 g of DHA/g of cell mass at the point of maximum conversion to 35.8 with oxygen enrichment. This increased specific production occurred even though the specific growth rate during the period of oxygen enrichment decreased from 0.23 to 0.06/h.     

Effect of oxygen dosing point and mixing on the microaerobic removal of hydrogen sulphide in sludge digesters

Limited oxygen supply to anaerobic sludge digesters to remove hydrogen sulphide from biogas was studied. Micro-oxygenation showed competitive performance to reduce considerably the additional equipment necessary to perform biogas desulphurization. Two pilot-plant digesters with an HRT of ∼20 d were micro-oxygenated at a rate of 0.25 NL per L of feed sludge with a removal efficiency higher than 98%. The way of mixing (sludge or biogas recirculation) and the point of oxygen supply (headspace or liquid phase) played an important role on hydrogen sulphide oxidation. While micro-oxygenation with sludge recirculation removed only hydrogen sulphide from the biogas, dissolved sulphide was removed if micro-oxygenation was performed with biogas recirculation. Dosage in the headspace resulted in a more stable operation. The result of the hydrogen sulphide oxidation was mostly elemental sulphur, partially accumulated in the headspace of the digester, where different sulphide-oxidising bacteria were found.     

Oxygen concentration profiles and exchange in sediment cores with circulated overlying water

SUMMARY. 1. The overlying water of intact sediment cores was constantly stirred with an impeller at a rate sufficient to mix turbulently the water column and maintain the diffusive boundary layer at a determined thickness. The system allowed standardization of water circulation in laboratory sediment core experiments.
2. Both oxygen concentration and oxygen penetration depth in the sediments decreased, the former by 70% and the latter from 4.2 mm to 2.0 mm, when the overlying water was not stirred for 24 h, as measured with oxygen microelectrodes in a lake sediment core.
3. Oxygen profiles measured in sediment cores in the laboratory were similar to those measured in situ when the overlying water was stirred with an impeller at such a rate that a similar thickness of the diffusive boundary layer at the sediment-water interface developed in the laboratory as that in situ.
4. Sediment oxygen consumption was calculated from: (1) measured oxygen profiles in the diffusive boundary layer and the molecular diffusion coefficient for oxygen in water; (2) the measured oxygen decrease in the top of the sediments and the estimated diffusion coefficient in the sediment; and (3) by oxygen differences in the overlying water after incubation of sediment cores.