Oxygen transfer and mixing in mechanically agitated airlift bioreactors

Gas holdup, mixing, liquid circulation and gas–liquid oxygen transfer were characterized in a large (∼1.5 m³) draft-tube airlift bioreactor agitated with Prochem^® hydrofoil impellers placed in the draft-tube. Measurements were made in water and in cellulose fiber slurries that resembled broths of mycelial microfungi. Use of mechanical agitation generally enhanced mixing performance and the oxygen transfer capability relative to when mechanical agitation was not used; however, the oxygen transfer efficiency was reduced by mechanical agitation. The overall volumetric gas–liquid mass transfer coefficient declined with the increasing concentration of the cellulose fiber solids; however, the mixing time in these strongly shear thinning slurries was independent of the solids contents (0–4% w/v). Surface aeration never contributed more than 12% to the total mass transfer in air–water.     

Impact of nozzle operation on mass transfer in jet aerated loop reactors. Characterization and comparison to an aerated stirred tank reactor

The impact of mass transfer on productivity can become a crucial aspect in the fermentative production of bulk chemicals. For highly aerobic bioprocesses the oxygen transfer rate (OTR) and productivity are coupled. The achievable space time yields can often be correlated to the mass transfer performance of the respective bioreactor. The oxygen mass transfer capability of a jet aerated loop reactor is discussed in terms of the volumetric oxygen mass transfer coefficient k_La [h^?1] and the energetic oxygen transfer efficiency E [kgO₂ kW^?1 h^?1]. The jet aerated loop reactor (JLR) is compared to the frequently deployed aerated stirred tank reactor. In jet aerated reactors high local power densities in the mixing zone allow higher mass transfer rates, compared to aerated stirred tank reactors. When both reactors are operated at identical volumetric power input and aeration rates, local k_La values up to 1.5 times higher are possible with the JLR. High dispersion efficiencies in the JLR can be maintained even if the nozzle is supplied with pressurized gas. For increased oxygen demands (above 120 mmol L^?1 h^?1) improved energetic oxygen transfer efficiencies of up to 100 % were found for a JLR compared to an aerated stirred tank reactor operating with Rushton turbines.     

Reprint of “Multiphase mixing characteristics in a microcarrier-based stirred tank bioreactor suitable for human mesenchymal stem cell expansion”

Large-scale human mesenchymal stem cell expansion calls for a bioreaction system, that provides a sufficient growth surface. An alternative to static cultivations systems like cell factories are disposable stirred tank reactors. Here, microcarriers provide the required growth surface, but these make it difficult to achieve a complete homogenization in the bioreactor, while avoiding shear stress. To gain insight into this process, we investigated the impact of different power inputs (0.02–2.6 W m⁻³) on the mixing time (t_m). Whereas t_m was inversely proportional to agitation in a one-phase-system, aeration resulted in a constant mixing time at 30–70 rpm. A high microcarrier concentration (30 g L⁻¹) and low stirrer speed (30 rpm) in the liquid-solid system caused a 50-fold increase in t_m and the formation of a discrete non-mixed upper zone. The effect of the microcarrier concentration on t_m became negligible at higher stirrer speeds. In the three-phase system, microcarrier settling was prevented by aeration and a minimal specific power input of 0.6 W m⁻³ was sufficient for complete homogenization. We confirmed that a low power input during stem cell expansion leads to inhomogeneity, which has not been investigated in the three-phase system up to date.     

Swainsonine,a novel fungal metabolite: optimization of fermentative production and bioreactor operations using evolutionary programming

The optimization of bioreactor operations towards swainsonine production was performed using an artificial neural network coupled evolutionary program (EP)-based optimization algorithm fitted with experimental one-factor-at-a-time (OFAT) results. The effects of varying agitation (300–500 rpm) and aeration (0.5–2.0 vvm) rates for different incubation hours (72–108 h) were evaluated in bench top bioreactor. Prominent scale-up parameters, gassed power per unit volume (P _g/V _L, W/m³) and volumetric oxygen mass transfer coefficient (K _L a, s^?1) were correlated with optimized conditions. A maximum of 6.59 ± 0.10 μg/mL of swainsonine production was observed at 400 rpm-1.5 vvm at 84 h in OFAT experiments with corresponding P _g/V_L and K _L a values of 91.66 W/m³ and 341.48 × 10^?4 s^?1, respectively. The EP optimization algorithm predicted a maximum of 10.08 μg/mL of swainsonine at 325.47 rpm, 1.99 vvm and 80.75 h against the experimental production of 7.93 ± 0.52 μg/mL at constant K _L a (349.25 × 10^?4 s^?1) and significantly reduced P _g/V _L (33.33 W/m³) drawn by the impellers.     

Scale-up and application of a cyclone reactor for fermentation processes

A cyclone reactor for microbial fermentation processes was developed with high oxygen transfer capabilities. Three geometrically similar cyclone reactors with 0.5?l, 2.5?l and 15?l liquid volume, respectively, were characterized with respect to oxygen mass transfer, mixing time and residence time distribution. Semi-empirically correlations for prediction of oxygen mass transfer and mixing times were identified for scale-up of cyclone reactors. A volumetric oxygen mass transfer coefficient k _L a of 1.0?s^?1 (available oxygen transfer rate with air: 29?kg?m^?3?h^?1) was achieved with the cyclone reactor at a volumetric power input of 40?kW?m^?3 and an aeration gas flow rate of 0.2?s^?1. Continuous methanol controlled production of formate dehydrogenase (FDH) with Candida boidinii in a 15?l cyclone reactor resulted in more than 100% improvement in dry cell mass concentration (64.5?g?l^?1) and in about 100% improvement in FDH space-time yield (300?U?l^?1?h^?1) compared to steady state results of a continuous stirred tank reactor.     

Hydrodynamic and Mass Transfer Characteristics of Three-Phase Gaslift Bioreactor Systems

ABSTRACT:?

This review focuses on the hydrodynamic and mass transfer characteristics of various three-phase, gaslift fluidized bioreactors. The factors affecting the mixing and volumetric mass transfer coefficient (k_La), such as liquid properties, solid particle properties, liquid circulation velocity, superficial gas velocity, bioreactor geometry, are reviewed and discussed. Measurement methods, modeling and empirical correlations are reviewed and compared. To the authors' knowledge, there is no 'generalized' correlation to calculate the volumetric mass transfer coefficient, instead, only 'type-specific' correlations are available in the literature. This is due to the difficulty in modeling the gaslift bioreactor, caused by the variation in geometry, fluid dynamics, and phase interactions. The most important design parameters reported in the literature are: gas hold-up, liquid circulation velocity, 'true' superficial gas velocity, mixing, shear rate, aeration rate and volumetric mass transfer coefficient, k_La.     

Selective fengycin production in a modified rotating discs bioreactor

Production of lipopeptides fengycin and surfactin in rotating discs bioreactor was studied. The effects of rotation velocity and the addition of agitators between the discs on volumetric oxygen transfer coefficient k _L a were firstly studied in model media. Then the production of lipopeptides was also studied at different agitation conditions in the modified bioreactor (with agitators). The effect of agitation on dissolved oxygen, on submerged and immobilized biomass, on lipopeptide concentrations and yields and on the selectivity of the bioreaction was elucidated and discussed. The proposed modified rotating discs bioreactor allowed to obtain high fengycin concentrations (up to 787 mg L^?1), but also better selectivity of the bioreaction towards fengycin (up to 88 %) and better yields of fengycin per glucose (up to 62.9 mg g^?1), lipopeptides per glucose (up to 71.5 mg g^?1), fengycin per biomass (up to 309 mg g^?1) and lipopeptides per biomass (up to 396 mg g^?1) than those reported in the literature. Highest fengycin production and selectivity were obtained at agitation velocity of 30 min^?1. The proposed non-foaming fermentation process could contribute to the scale-up of lipopeptide fermentors and promote the industrial production of fengycin. The proposed bioreactor and bioprocess could be very useful also for the production of other molecules using bioprocesses requiring bubbleless oxygen supply.     

Development of a bioreactor for high density culture of hairy roots

Summary The effects of agitation and aeration on the growth of carrot hairy roots were investigated. When hydrodynamic stress index was above 0.001 cm/s, the growth rate of hairy roots decreased sharply. When volumetric O₂ transfer coefficient was high, the specific growth rate was also high. However, the specific growth rate approached the maximum value when the volumetric O₂ transfer coefficient was over 4 h^–1 . It is therefore necessary to maintain low hydrodynamic stress and high volumetric oxygen transfer for high density culture of hairy roots. By considering hydrodynamic stress and oxygen transfer, a novel bioreactor type was suggested for hairy roots cultivation.This research was supported in part by the Genetic Engineering Research Fund Korean Ministry of Education     

Production and characterization of Penicillium brasilianum pectinases with regard to industrial application

The objective of this study was to evaluate the production of pectinase by an isolated strain of Penicillium brasilianum in a bioreactor and to consider its potential for industrial applications (i.e. fruit juice). The optimization of production was achieved through experimental design. The maximum exo-polygalacturonase (Exo-PG) production in the bioreactor was 53.8?U mL^?1 under the conditions of 180?rpm, an aeration rate of 1.5 vvm, 30?°C, pH_initial of 5.5, 5?×?10⁶ spores mL^?1, 32?g L^?1 pectin, 10?g L^?1 of yeast extract and 0.5?g L^?1 magnesium sulfate and bioproduction for 36?h. The production of Exo-PG in the bioreactor was 1.3 times higher than that obtained in shake flasks, with aeration (1.5 vvm) and agitation (180?rpm) control. The crude enzyme complex, beyond the pectinolytic activity of Exo-PG (53.8?U mL^?1), also contained activity pectin methylesterase (6.0?U mL^?1) and pectin lyase (6.61?U mL^?1). At a crude enzyme complex with a concentration of 0.5% (v/v), viscosity of peach juice was reduced by 11.66%, turbidity was reduced by 13.71% and clarification was increased by 26.92%. Based on the present results, we can conclude that the new strain of isolated P. brasilianum produced high amounts of pectinases in a bioreactor with mechanical agitation, and has the potential to be applied to in the clarification of juices.     

Transfer of an adherent Vero cell culture method between two different rocking motion type bioreactors with respect to cell growth and metabolic rates

Rapid and simple cell and virus cultivation can currently be carried out using disposable bioreactors. The CELL-tainer^® (CELLution Biotech BV) disposable bioreactor is a rocking-type bioreactor which not only has vertical movement but horizontal displacement as well. Due to this two-directional movement relatively high mass-transfer capacities can be reached when compared with conventional rocking motion-type bioreactors.Using the design of experiments (DoE) approach we have developed models for the mixing times in both the CELL-tainer^® and the BIOSTAT^® CultiBag RM (Sartorius Stedim Biotech) bioreactor (standard rocking motion-type). The conditions for cultivation of Vero cells in the CELL-tainer^® bioreactor were chosen based on comparable mixing times.Vero cells growing adherent to Cytodex 1 microcarriers were cultivated in the CELL-tainer^® and in the BIOSTAT^® CultiBag RM. Both bioreactors were controlled with regard to temperature, pH and % dissolved oxygen. Vero cell growth in both bioreactors was comparable with respect to the growth characteristics and main metabolite production and consumption rates. Additionally, polio virus production in both bioreactors was shown to be similar.     

Scale-up from shake flasks to bioreactor, based on power input and Streptomyces lividans morphology, for the production of recombinant APA (45/47 kDa protein) from Mycobacterium tuberculosis

Culture conditions in shake flasks affect filamentous Streptomyces lividans morphology, as well the productivity and O-mannosylation of recombinant Ala-Pro-rich O-glycoprotein (known as the 45/47 kDa or APA antigen) from Mycobacterium tuberculosis. In order to scale up from previous reported shake flasks to bioreactor, data from the literature on the effect of agitation on morphology of Streptomyces strains were used to obtain gassed volumetric power input values that can be used to obtain a morphology of S. lividans in bioreactor similar to the morphology previously reported in coiled/baffled shake flasks by our group. Morphology of S. lividans was successfully scaled-up, obtaining similar mycelial sizes in both scales with diameters of 0.21 ± 0.09 mm in baffled and coiled shake flasks, and 0.15 ± 0.01 mm in the bioreactor. Moreover, the specific growth rate was successfully scaled up (0.09 ± 0.02 and 0.12 ± 0.01 h^?1, for bioreactors and flasks, respectively), and the recombinant protein productivity measured by densitometry, as well. More interestingly, the quality of the recombinant glycoprotein measured as the amount of mannoses attached to the C-terminal of APA was also scaled- up; with up to five mannose residues in cultures carried out in shake flasks; and six in the bioreactor. However, final biomass concentration was not similar, indicating that although the process can be scaled-up using the power input, others factors like oxygen transfer rate, tip speed or energy dissipation/circulation function can be an influence on bacterial metabolism.     

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.     

Production of influenza H1N1 vaccine from MDCK cells using a novel disposable packed-bed bioreactor

A process for human influenza H1N1 virus vaccine production from Madin–Darby canine kidney (MDCK) cells using a novel packed-bed bioreactor is described in this report. The mini-bioreactor was used to study the relationship between cell density and glucose consumption rate and to optimize the infection parameters of the influenza H1N1 virus (A/New Caledonia/20/99). The MDCK cell culture and virus infection were then monitored in a disposable perfusion bioreactor (AmProtein Current Perfusion Bioreactor) with proportional–integral–derivative control of pH, dissolved O₂ (DO), agitation, and temperature. During 6 days of culture, the total cell number increased from 2.0?×?10⁹ to 3.2?×?10¹⁰ cells. The maximum virus titers of 768 hemagglutinin units/100 μL and 7.8?×?10⁷ 50 % tissue culture infectious doses/mL were obtained 3 days after infection. These results demonstrate that using a disposable perfusion bioreactor for large-scale cultivation of MDCK cells, which allows for the control of DO, pH, and other conditions, is a convenient and stable platform for industrial-scale production of influenza vaccines.     

Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield

Streptavidin is widely used as an analytical tool and affinity tag together with biotinylated surfaces or molecules. We report for the first time a simple strategy that yields high biomass of a Pichia pastoris strain containing a methanol induced core streptavidin (cStp) gene. Three factors were evaluated for biomass production: glycerol concentration, aeration, and feed flow rates in a bioreactor. Recycling of recombinant cells, either free or immobilized, was investigated during induction. Concentration of 2.0 M glycerol, feeding flow rate of 0.11 mL min^?1, and aeration by air injection dispersed with a porous stone combined with agitation at 500 rpm were the set of conditions resulting into maximum biomass yield (150 g L^?1). These parameters yielded 4.0 g L^?1 of cStp, after 96 h of induction. Recombinant biomass was recycled twice before being discarded, which can reduce production costs and simplify the process. Immobilized P. pastoris biomass produced 2.94 and 1.70 g L^?1 of cStp in the first and second induction cycle, respectively. Immobilization and recycling of recombinant P. pastoris biomass opens new possibilities as a potential strategy to improve volumetric productivity for heterologous protein expression. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Optimization of production of caffeine demethylase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. in a bioreactor

The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight⁻¹ h⁻¹ was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h⁻¹, indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors.     

Two new disposable bioreactors for plant cell culture: The wave and undertow bioreactor and the slug bubble bioreactor

The present article describes two novel flexible plastic-based disposable bioreactors. The first one, the WU bioreactor, is based on the principle of a wave and undertow mechanism that provides agitation while offering convenient mixing and aeration to the plant cell culture contained within the bioreactor. The second one is a high aspect ratio bubble column bioreactor, where agitation and aeration are achieved through the intermittent generation of large diameter bubbles, "Taylor-like" or "slug bubbles" (SB bioreactor). It allows an easy volume increase from a few liters to larger volumes up to several hundred liters with the use of multiple units. The cultivation of tobacco and soya cells producing isoflavones is described up to 70 and 100 L working volume for the SB bioreactor and WU bioreactor, respectively. The bioreactors being disposable and pre-sterilized before use, cleaning, sterilization, and maintenance operations are strongly reduced or eliminated. Both bioreactors represent efficient and low cost cell culture systems, applicable to various cell cultures at small and medium scale, complementary to traditional stainless-steel bioreactors.     

Establishing the scalable manufacture of primary human T-cells in an automated stirred-tank bioreactor

Advanced cell and gene therapies such as chimeric antigen receptor T-cell immunotherapies (CAR-T), present a novel therapeutic modality for the treatment of acute and chronic conditions including acute lymphoblastic leukemia and non-Hodgkin lymphoma. However, the development of such immunotherapies requires the manufacture of large numbers of T-cells, which remains a major translational and commercial bottleneck due to the manual, small-scale, and often static culturing systems used for their production. Such systems are used because there is an unsubstantiated concern that primary T-cells are shear sensitive, or prefer static conditions, and therefore do not grow as effectively in more scalable, agitated systems, such as stirred-tank bioreactors, as compared with T-flasks and culture bags. In this study, we demonstrate that not only T-cells can be cultivated in an automated stirred-tank bioreactor system (ambr® 250), but that their growth is consistently and significantly better than that in T-flask static culture, with equivalent cell quality. Moreover, we demonstrate that at progressively higher agitation rates over the range studied here, and thereby, higher specific power inputs (P/M W kg⁻¹), the higher the final viable T-cell density; that is, a cell density of 4.65 ± 0.24 × 10⁶ viable cells ml⁻¹ obtained at the highest P/M of 74 × 10⁻⁴ W kg⁻¹ in comparison with 0.91 ± 0.07 × 10⁶ viable cells ml⁻¹ at the lowest P/M of 3.1 × 10⁻⁴ W kg⁻¹. We posit that this improvement is due to the inability at the lower agitation rates to effectively suspend the Dynabeads®, which are required to activate the T-cells; and that contact between them is improved at the higher agitation rates. Importantly, from the data obtained, there is no indication that T-cells prefer being grown under static conditions or are sensitive to fluid dynamic stresses within a stirred-tank bioreactor system at the agitation speeds investigated. Indeed, the opposite has proven to be the case, whereby, the cells grow better under higher agitation speeds while maintaining their quality. This study is the first demonstration of primary T-cell ex vivo manufacture activated by Dynabeads® in an automated stirred-tank bioreactor system such as the ambr® 250 and the findings have the potential to be applied to multiple other cell candidates for advanced therapy applications.     

Determination of specific oxygen uptake rate of Photorhabdus luminescens during submerged culture in lab scale bioreactor

Photorhabdus luminescens, a bacterial symbiont of entomoparasitic nematodes, was cultured in a 10 L bioreactor. Cellular density and bioluminescence were recorded and volumetric oxygen transfer coefficient (k_La) and specific oxygen transfer rates were determined during the batch process. Exponential phase of the bacterium lasted for 20 h, showing a maximum specific growth rate of 0.339 h^?1 in a defined medium. Bioluminescence peaked within 21h, and was maintained until the end of the batch process (48 h). The specific oxygen uptake rate (SOUR) was high during both lag and early exponential phase, and eventually reached a stable value of 0.33 mmol g^?1 h^?1 during stationary phase. Maintenance of 200 rpm agitation and 1.4 volume of air per volume of medium per minute (vvm) aeration, gave rise to a k_La value of 39.5 h^?1. This k_La value was sufficient to meet the oxygen demand of 14.4 g L^?1 (DCW) biomass. This research is particularly relevant since there are no reports available on SOURs of symbiotic bacteria or their nematode partners. The insight gained through this study will be useful during the development of a submerged monoxenic culture of Heterorhabditis bacteriophora and its symbiotic bacterium P. luminescens in bioreactors.     

Scale‐up analysis for a CHO cell culture process in large‐scale bioreactors

Bioprocess scale‐up is a fundamental component of process development in the biotechnology industry. When scaling up a mammalian cell culture process, it is important to consider factors such as mixing time, oxygen transfer, and carbon dioxide removal. In this study, cell‐free mixing studies were performed in production scale 5,000‐L bioreactors to evaluate scale‐up issues. Using the current bioreactor configuration, the 5,000‐L bioreactor had a lower oxygen transfer coefficient, longer mixing time, and lower carbon dioxide removal rate than that was observed in bench scale 5‐ and 20‐L bioreactors. The oxygen transfer threshold analysis indicates that the current 5,000‐L configuration can only support a maximum viable cell density of 7 × 10⁶ cells mL^?1. Moreover, experiments using a dual probe technique demonstrated that pH and dissolved oxygen gradients may exist in 5,000‐L bioreactors using the current configuration. Empirical equations were developed to predict mixing time, oxygen transfer coefficient, and carbon dioxide removal rate under different mixing‐related engineering parameters in the 5,000‐L bioreactors. These equations indicate that increasing bottom air sparging rate is more efficient than increasing power input in improving oxygen transfer and carbon dioxide removal. Furthermore, as the liquid volume increases in a production bioreactor operated in fed‐batch mode, bulk mixing becomes a challenge. The mixing studies suggest that the engineering parameters related to bulk mixing and carbon dioxide removal in the 5,000‐L bioreactors may need optimizing to mitigate the risk of different performance upon process scale‐up. Biotechnol. Bioeng. 2009;103: 733–746. © 2009 Wiley Periodicals, Inc.     

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.