Fluid mixing and oxygen transfer in cell suspensions ofTaxus chinensis in a novel stirred bioreactor

In high-density plant cell cultures, mixing and mass transfer are two key issues, which should be emphasized for process optimization. In this work, both mixing and oxygen transfer characteristics of cell suspensions ofTaxus chinensis were studied in a new centrifugal impeller bioreactor with a working volume of 1.2 L. The mixing time (t _M) and the volumetric oxygen transfer coefficient (K _L a) under different operational conditions were determined in both tap water and cell suspensions of 100–400 g fresh weight/L (i.e., 5.65–23.1 g DW/L). At an aeration rate of 0.1 L/min,t _M decreased from 10.6s at 30 rpm to 2.89 s at 200 rpm under 100 g FW/L, and from 9.63 s (120 rpm) to 4.05 s (300 rpm) under 400 g FW/L. Compared with the effect of agitation, aeration was less significant to the suspension mixing. At a relatively high agitation speed (e.g., 200 rpm),t _M remained almost the same even though aeration rate was changed from 0.1 to 0.4 L/min. Thet _M value increased slowly from 3.98 to 5.26 s at 120 rpm when the cell density was raised from 100 to 250 g FW/L. A rapid increase of botht _M and the suspension viscosity was observed at a cell density above 300 g FW/L. As expected, theK _L a value increased with an increase of aeration rate and agitation speed, but decreased with an increase of cell density. The quantitative data obtained here are useful to investigate the effect of mixing stress on the cell physiology and metabolism ofTaxus chinensis in the bioreactor. This paper is dedicated by JJZ to his colleague Prof. Jun-Tang Yu on the occasion of his 70 birthday.     

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 In the proposed system [2] the riser has been replaced by a tube of irregular geometry, in the form of converging-diverging sections (CDT-ALF), so that better mass transfer may be obtained due to better liquid mixing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     

Influence of agitation and aeration on growth and antibiotic production by <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

The effect of agitation and aeration on the growth and antibiotic production by Xenorhabdus nematophila YL001 grown in batch cultures were investigated. Efficiency of aeration and agitation was evaluated through the oxygen mass transfer coefficient (K _L a). With increase in K _L a, the biomass and antibiotic activity increased. Activity units of antibiotic and dry cell weight were increased to 232 U ml⁻¹ and 19.58 g l⁻¹, respectively, productivity in cell and antibiotic was up more than 30% when K _L a increased from 115.9 h⁻¹ to 185.7 h⁻¹. During the exponential growth phase, DO concentration was zero, the oxygen supply was not sufficient. So, based on process analysis, a three-stage oxygen supply control strategy was used to improved the DO concentration above 30% by controlling the agitation speed and aeration rate. The dry cell weight and activity units of antibiotic were further increased to 24.22 g l⁻¹ and 249 U ml⁻¹, and were improved by 24.0% and 7.0%, compared with fermentation at a constant agitation speed and a constant aeration rate (300 rev min⁻¹, 2.5 l min⁻¹).     

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.     

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.     

The influence of geometry on hydrodynamic and mass transfer characteristics in an external airlift reactor for the cultivation of filamentous fungi

The influences of geometric configuration, mycelial broth rheology and superficial gas velocity (U_sg) were investigated with respect to the following hydrodynamic parameters: gas holdup (), oxygen transfer coefficient (K_La) and mixing time (t_m). Increases in U_sg and height of gas separator (H_t) caused an increase in and K_La, and a decrease in t_m. Consequently, a diameter ratio (D_d/D_r) of 0.71 and H_t 0.20 m were found to be the best geometry and operation parameters to achieve high aeration and mixing efficiency for the high viscous broth system in the cultivation of filamentous fungi. An external airlift reactor (EALR) was developed and designed for the cultivation of filamentous fungi. The EALR with two spargers excels in reliability and high aeration and mass transfer coefficiency, resulting in a fast mycelial growth and high biomass productivity in the cultivation of the fungus Rhizopus oryzae.     

High density cultivation of plant cells in a new aeration-agitation type fermentor,maxblend fermentor®

The applicability of a new aeration-agitation type fermentor with a grid-paddle type impeller and a spiral-sparger, Maxblend Fermentor® (MBF) for high density cultivation of plant cells, was investigated. The MBF showed a high capacity for oxygen supply and extremely low hydrodynamic stress in aeration and mixing compared with a conventional fermentor (CF). When Oryza sativa cells were cultivated at a k_La of 20 h⁻¹, a high cell density cultivation of about 30 g dry cell weight per liter was accomplished in both fermentors and there were few differences in culture performance between the two. On the contrary, considerable differences were observed when Catharanthus roseus cells, which seemed to be sensitive to physical stress, were cultivated at a k_La of 20 h⁻¹ in both fermentors. The MBF exhibited excellent cell growth characteristics, achieving about 19 g dry cell weight per liter, because of its superior oxygen supply and low hydrodynamic stress in aeration and mixing in highly viscous cultures containing high density cells. In CF only about 9.5 g dry cell weight per liter was achieved because of its high hydrodynamic stress.     

Enhanced in situ dynamic method for measuring KLa in fermentation media

The overall oxygen mass transfer coefficient (K_La) is often used as scale-up factor of fermentation systems. In fermenter scale-up, it is desired to achieve the same K_La values at the larger scale than the one that was obtained at a smaller scale during the development stage. It is therefore important to be able to measure K_La in situ during fermentation and to also determine the action to be taken to maintain its value at its design set point. These objectives can be obtained by measuring K_La using the dynamic method and enhancing the K_La information by immediately conducting a series of changes in agitation speed and/or aeration rate to determine the influence of these variables on K_La. This enhanced dynamic method is demonstrated with two filamentous microorganisms: Trichoderma reesei for the production of cellulase and Aspergillus niger for the production of citric acid. Two different types of bioreactor were used: a reciprocating plate bioreactor and a stirred (Rushton) bioreactor. It is shown that the proposed method can provide a simple way to measure the local variation of K_La and to adjust its value to its set point during the course of fermentation.     

Performance intensification of a stirred bioreactor for fermentative biohydrogen production

In this study, the biohydrogen (bioH₂) production of a microbial consortium was optimized by adjusting the type and configuration of two impellers, the mixing regimen and the mass transfer process (K_La coefficients). A continuous stirred-tank reactor (CSTR) system, with a nonstandard geometry, was characterized. Two different mixing configurations with either predominant axial (PB4 impeller) or radial pumping (Rushton impeller) were assessed and four different impeller configurations to produce bioH₂. The best configuration for an adequate mixing time was determined by an ANOVA analysis. A response surface methodology was also used to fully elucidate the optimal configuration. When the PB4 impellers were placed in best configuration, c/Dt?=?0.5, s/Di?=?1, the maximum bioH₂ productivity obtained was 440?mL?L^?1?hr^?1, with a bioH₂ molar yield of 1.8. The second best configuration obtained with the PB4 impellers presented a bioH₂ productivity of 407.94?mL?L^?1?hr^?1. The configurations based on Rushton impellers showed a lower bioH₂ productivity and bioH₂ molar yield of 177.065?mL?L^?1?hr^?1 and 0.71, respectively. The experiments with axial impellers (PB4) showed the lowest K_La coefficient and the highest bioH₂ production, suggesting that mixing is more important than K_La for the enhanced production of bioH_2.     

Effect of fermentation variables on cellulase production by Trichoderma Sp.

Summary Batch cultivation ofTrichodermma reesei QM9414 was carried out in Mandels medium containing(w/v) 1% beech wood cellulose and 0.05% yeast extract at 29°C. Use of 36 hours old inoculum(10% v/v),3.2 1/min aeration rate at 400 rpm(K_La 220/h) and pH cycling strategy produced 4 g/1 cell mass and 21.5 IU/1/h FPA cellulase.     

Comparative investigation of fine bubble and macrobubble aeration on gas utility and biotransformation productivity

The sufficient provision of oxygen is mandatory for enzymatic oxidations in aqueous solution, however, in process optimization this still is a bottleneck that cannot be overcome with the established methods of macrobubble aeration. Providing higher mass transfer performance through microbubble aerators, inefficient aeration can be overcome or improved. Investigating the mass transport performance in a model protein solution, the microbubble aeration results in higher k_La values related to the applied airstream in comparison with macrobubble aeration. Comparing the aerators at identical k_La of 160 and 60 1/h, the microbubble aeration is resulting in 25 and 44 times enhanced gas utility compared with aeration with macrobubbles. To prove the feasibility of microbubbles in biocatalysis, the productivity of a glucose oxidase catalyzed biotransformation is compared with macrobubble aeration as well as the gas‐saving potential. In contrast to the expectation that the same productivities are achieved at identically applied k_La, microbubble aeration increased the gluconic acid productivity by 32% and resulted in 41.6 times higher oxygen utilization. The observed advantages of microbubble aeration are based on the large volume‐specific interfacial area combined with a prolonged residence time, which results in a high mass transfer performance, less enzyme deactivation by foam formation, and reduced gas consumption. This makes microbubble aerators favorable for application in biocatalysis.     

A neural network model for estimating O2 absorption coefficient in shake-flasks

A comprehensive, simple, neural network model was constructed to replace the common semi-empirical mathematical models used for predicting individual O₂ absorption coefficients (K _L a) within Erlenmeyer and Hinton shake-flasks. Different factors that influence K _L a within shake-flasks, such as flask dimensions, working volumes, baffle-heights, and shaking speeds, were investigated and the experimental results employed to deduce the mathematical model for each type of shake-flask. Meanwhile, the K _L a values calculated from the mathematical models were used to derive a non-linear neural network estimator (NNE). The NNE for K _L a prediction was implemented to evaluate the O₂ absorption effect within the flasks and gave a promising result.     

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.     

kLa of stirred tank bioreactors revisited

By means of improved feedback control k_La measurements become possible at a precision and reproducibility that now allow a closer look at the influences of power input and aeration rate on the oxygen mass transfer. These measurements are performed online during running fermentations without a notable impact on the biochemical conversion processes. A closer inspection of the mass transfer during cultivations showed that at least the number of impellers influences mass transfer and mixing: On the laboratory scale, two hollow blade impellers clearly showed a larger k_La than the usually employed three impeller versions when operated at the same agitation power and aeration rate. Hollow blade impellers are preferable under most operational conditions because of their perfect gas handling capacity. Mixing time studies showed that these two impeller systems are also preferable with respect to mixing. Furthermore the widths of the baffle bars depict a significant influence on the k_La. All this clearly supports the fact that it is not only the integral power density that finally determines k_La.     

Carbon dioxide absorption characterization of a bioreactor for biomass production ofPhormidium bohneri: comparative study of three types of diffuser

Under both laboratory-controlled and outdoor conditions, mass culture ofPhormidium bohneri has shown low growth rates and filament aggregate or floc instability. In order to test for a possible carbonaceous supply limitation, a mass transfer characterization study of the reactor used was conducted. To examine different conditions of aeration, the overall volumetric mass transfer coefficient, K_La(C0₂), and the energy and physical transfer efficiency for CO₂ were determined for three air-flow rates and three types of diffuser. For the different aeration conditions studied, the reactor showed adequate mixing properties with respect to CO₂. However, under low air-flow rate conditions, even with the most efficient diffuser, the CO₂ transfer capacity appeared limiting. On the other hand, at high air-flow rates, floc breakage as a result of shear stress appeared to limit bioreactor efficiency.     

Oxygen transfer in mechanically agitated aqueous systems containing dispersed hydrocarbon

The effect of dispersed n -dodecane or n -hexadecane on the air-to-aqueous phase overall volumetric oxygen transfer coefficient in a simulated (cell-free) stirred-tank fermentor is described. The oil volume fraction ranged from zero to 0.10; the ionic strength of the aqueous phases was varied from 0 to 0.45. The air-to-aqueous phase coefficients in both oil-free (K_La) and oil-bearing (K_La*) systems were evaluated from unsteady-state experiments using a membrane-covered probe to follow the aqueous phase dissolved oxygen tension. For all systems studied, K_La*/K_La was found to be independent of P/V and v_s for all practical purposes. However, for a particular aqueous phase and at a given P/V and v_s, the ratio K_La*K_La generally differed from unity. Depending on the combination of hydrocarbon type and volume fraction and the aqueous-phase ionic strength employed, the dispersed hydrocarbon may, in some cases, reduce the rate of oxygen transfer and in others enhance it relative to that of the corresponding oil-free gas–liquid dispersion. Enhancement of the air-to-aqueous transfer rate by such negative spreading coefficient hydrocarbons has not been reported previously.     

Voltage dependence of the rheogenic Na+/K+ ATPase in the membrane of oocytes ofXenopus laevis

Summary Electrophysiological experiments were performed to analyze the Na⁺/K⁺-ATPase in full-grown prophase-arrested oocytes ofXenopus laevis. If the Na⁺/K⁺-ATPase is inhibited by dihydroouabain (DHO), the resting potential of the membrane of Na⁺-loaded oocytes may depolarize by nearly 50 mV. This hyperpolarizing contribution to the resting potential depends on the degree of activation of the Na⁺/K⁺-ATPase and varies with intra-cellular Na⁺ activity (a _Na ⁱ ), and extracellular K⁺ (K ₀ ⁺ ) It is concluded that variations ofa _Na ⁱ among different oocytes are primarily responsible for the variations of resting potentials measured in oocytes ofX. laevis. Under voltage-clamp conditions, the DHO-sensitive current also exhibits dependence ona _Na ⁱ that may be described by a Hill equation with a coefficient of 2. This current will be shown to be identical with the electrogenic current generated by the 3Na⁺/2K⁺ pump. The voltage dependence of the pump current was investigated at saturating values ofa _Na ⁱ (33 mmol/liter) and of K ₀ ⁺ (3 mmol/liter) in the range from –200 to +100 mV. The current was found to exhibit a characteristic maximum at about +20 mV. This is taken as evidence that in the physiological range at least two steps within the cycle of the pump are voltage dependent and are oppositely affected by the membrane potential.     

Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: mathematical modeling

The effect of aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor was investigated. B. trispora formed hyphae, zygophores and zygospores during the fermentation. The zygospores were the morphological form responsible for β-carotene production. Both aeration and agitation significantly affected β-carotene concentration, productivity, biomass and the volumetric mass transfer coefficient (K_La). The highest β-carotene concentration (1.5 kg m⁻³) and the highest productivity (0.08 kg m⁻³ per day) were obtained at low impeller speed (150 rpm) and high aeration rate (1.5 vvm). Also, maximum productivity (0.08 kg m⁻³ per day) and biomass dry weight (26.4 kg m⁻³) were achieved at high agitation speed (500 rpm) and moderate aeration rate (1.0 vvm). Conversely, the highest value of K_La (0.33 s⁻¹) was observed at high agitation speed (500 rpm) and high aeration rate (1.5 vvm). The experiments were arranged according to a central composite statistical design. Response surface methodology was used to describe the effect of impeller speed and aeration rate on the most important fermentation parameters. In all cases, the fit of the model was found to be good. All fermentation parameters (except biomass concentration) were strongly affected by the interactions among the operation variables. β-Carotene concentration and productivity were significantly influenced by the aeration, agitation, and by the positive or negative quadratic effect of the aeration rate. Biomass concentration was principally related to the aeration rate, agitation speed, and the positive or negative quadratic effect of the impeller speed and aeration rate, respectively. Finally, the volumetric mass transfer coefficient was characterized by the significant effect of the agitation speed, while the aeration rate had a small effect on K_La.     

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.     

A case study in converting disposable process scouting devices into disposable bioreactors as a future bioprocessing tool

In this study, we perform mass transfer characterization (k_La) on a novel mechanically driven/stirred Process Scouting Device, PSD, (SuperSpinner D 1000®, SSD) and demonstrate that this novel device can be viewed as disposable bioreactor. Using patch‐based optical sensors, we were able to monitor critical cell culture environmental conditions such as dissolved oxygen (DO) and pH in SSD for comparison to a 1 L standard spinner (SS) flask. We also coupled these mass transfer studies with mixing time studies where we observed relative high mixing times (5.2 min) that are typically observed in production scale bioreactors. Decreasing the mixing time 3.5‐fold resulted in 30% increase in k_La (from 2.3 to 3.0 h^?1) and minimum DO level increased from 0% to 20% for our model hybridoma cell line. Finally, maximum viable cell density and protein titer stayed within ±20% of historical data, from our standard 5 L stirred bioreactor (Biostat®) operated under active DO control. Biotechnol. Bioeng. 2012; 109: 2790–2797. © 2012 Wiley Periodicals, Inc.