Studies on fluid dynamics of the flow field and gas transfer in orbitally shaken tubes

Orbitally shaken cylindrical bioreactors [OrbShake bioreactors (OSRs)] without an impeller or sparger are increasingly being used for the suspension cultivation of mammalian cells. Among small volume OSRs, 50‐mL tubes with a ventilated cap (OSR50), originally derived from standard laboratory centrifuge tubes with a conical bottom, have found many applications including high‐throughput screening for the optimization of cell cultivation conditions. To better understand the fluid dynamics and gas transfer rates at the liquid surface in OSR50, we established a three‐dimensional simulation model of the unsteady liquid forms (waves) in this vessel. The studies verified that the operating conditions have a large effect on the interfacial surface. The volumetric mass transfer coefficient (k_La) was determined experimentally and from simulations under various working conditions. We also determined the liquid‐phase mass transfer coefficient (k_L) and the specific interfacial area (a) under different conditions to demonstrate that the value of a affected the gas transfer rate more than did the value of k_L. High oxygen transfer rates, sufficient for supporting the high‐density culture of mammalian cells, were found. Finally, the average axial velocity of the liquid was identified to be an important parameter for maintaining cells in suspension. Overall these studies provide valuable insights into the preferable operating conditions for the OSR50, such as those needed for cell cultures requiring high oxygen levels. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:192–200, 2017     

Time efficient way to calculate oxygen transfer areas and power input in cylindrical disposable shaken bioreactors

Disposable orbitally shaken bioreactors are a promising alternative to stirred or wave agitated systems for mammalian and plant cell cultivation, because they provide a homogeneous and well‐defined liquid distribution together with a simple and cost‐efficient design. Cultivation conditions in the surface‐aerated bioreactors are mainly affected by the size of the volumetric oxygen transfer area (a) and the volumetric power input (P∕V_L) that both result from the liquid distribution during shaking. Since Computational Fluid Dynamics (CFD)—commonly applied to simulate the liquid distribution in such bioreactors—needs high computing power, this technique is poorly suited to investigate the influence of many different operating conditions in various scales. Thus, the aim of this paper is to introduce a new mathematical model for calculating the values of a and P∕V_L for liquids with water‐like viscosities. The model equations were derived from the balance of centrifugal and gravitational forces exerted during shaking. A good agreement was found among calculated values for a and P∕V_L, CFD simulation values and empirical results. The newly proposed model enables a time efficient way to calculate the oxygen transfer areas and power input for various shaking frequencies, filling volumes and shaking and reactor diameters. All these parameters can be calculated fast and with little computing power. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1441–1456, 2014     

Oxygen transfer in slurry bioreactors

The oxygen transfer in bioreactors with slurries having a yield stress was investigated. The volumetric mass transfer coefficients in a 40-L bubble column with simulated fermentation broths, the Theological properties of which were represented by the Casson model, were measured. Experimental data were compared with a theoretical correlation developed on the basis of a combination of Higbie's penetration theory and Kolmogoroff's theory of isotropic turbulence. Comparisons between the proposed correlation and data for the simulated broths show good agreement. The mass transfer data for actual mycelial fermentation broths reported previously by the authors were re-examined. Their Theological data was correlated by the Bingham plastic model. The oxygen transfer rate data in the mycelial fermentation broths fit the predictions of the proposed theoretical correlation.     

Over‐pressurized bioreactors: Application to microbial cell cultures

In industrial biotechnology, microbial cultures are exposed to different local pressures inside bioreactors. Depending on the microbial species and strains, the increased pressure may have detrimental or beneficial effects on cellular growth and product formation. In this review, the effects of increased air pressure on various microbial cultures growing in bioreactors under moderate total pressure conditions (maximum, 15 bar) will be discussed. Recent data illustrating the diversity of increased air pressure effects at different levels in microbial cells cultivation will be presented, with particular attention to the effects of oxygen and carbon dioxide partial pressures on cellular growth and product formation, and the concomitant effect of oxygen pressure on antioxidant cellular defense mechanisms. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:767–775, 2014     

Oxygen transfer deficiencies in starch-based media

Insufficient oxygen transfer in a highly viscous media with varying viscosity occurs during the degradation of starch byBacillus licheniformis. Oxygen transfer rate (OTR) decreased below 0.3 gl^–1 h^–1 for viscosities above 5 to 10 mPa.s and agitation speeds lower than 710 rev/min. Increasing agitation speeds by 30 to 50% compensated for the decrease in OTR for viscosities between 10 and 200 mPa.s. A dual impeller is considered essential for growth improvement.     

Influence of pluronic F68 on oxygen mass transfer

Pluronic F68 is one of the most used shear protecting additives in cell culture cultivations. It is well known from literature that such surface‐active surfactants lower the surface tension at the gas‐liquid interface, which influences the mass transfer. In this study, the effect of Pluronic F68 on oxygen mass transfer in aqueous solutions was examined. Therefore, the gassing in/gassing out method and bubble size measurements were used. At low concentrations of 0.02 g/L, a 50% reduction on mass transfer was observed for all tested spargers and working conditions. An explanation of the observed effects by means of Higbie's penetration or Dankwerts surface renewal theory was applied. It could be demonstrated that the suppressed movement of the bubble surface layer is the main cause for the significant drop down of the k_La‐values. For Pluronic F68 concentrations above 0.1 g/L, it was observed that it comes to changes in bubble appearance and bubble size strongly dependent on the sparger type. By using the bubble size measurement data, it could be shown that only small changes in mass transfer coefficient (k_L) take place above the critical micelle concentration. Further changes on overall mass transfer at higher Pluronic F68 concentrations are mainly based on increasing of gas holdup and, more importantly, by increasing of the surface area available for mass transfer. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1278–1288, 2013     

Effect of dissolved oxygen concentration on differentiation of somatic embryos of Coffea arabica cv. Catimor 9722

The effect of two different dissolved oxygen (DO) concentrations (50 and 80%) on differentiation of somatic embryos (SE) from cell suspensions of coffee (Coffea arabica cv. Catimor 9722) was analyzed. Two bioreactors CMF-100 (CHEMAP AG) designed for the culture of cells, with 2-l glass vessels and a maximum work volume of 1.8 l were used. Each one was equipped with a gas blending unit (air, O₂, N₂, CO₂) for the control of DO concentration. The inoculation density of embryogenic cells was 1.0 gram of fresh weight per liter (g FW l^–1). The number of somatic embryos was greater (71 072 SE l^–1) with 80% DO, but the major proportion were globular and heart shaped SE (66 399 SE l^–1) and only 6.6% with regard to total was torpedo shaped SE. However, the 50% DO produced the higher number in the torpedo shaped SE (7389 SE l^–1) what represented 20.0% with regard to total. Thus, higher concentrations of DO induced globular and heart shaped SE differentiation, but for production of torpedo shaped SE lower concentrations DO are needed. The somatic embryos obtained in the bioreactor with 50% DO showed similar behavior to the somatic embryos obtained in the rotary shaker. After 8 weeks of culture, 49.2% germination was obtained, which allowed a total of 1725 plantlet to be transferred to conditions ex vitro. After 6 months of culture, 89.2% of conversion was achieved and 1539 plants obtained were transferred to field conditions.     

Oxygen transfer effects in serine alkaline protease fermentation by Bacillus licheniformis: use of citric acid as the carbon source

The effects of oxygen transfer on serine alkaline protease (SAP) production by Bacillus licheniformis on a defined medium with C_c = 9.0 kg m⁻³ citric acid as sole carbon source were investigated in 3.5 dm³ batch bioreactor systems. The concentrations of the product (SAP) and by-products, i.e., neutral protease, amylase, amino acids, and organic acids were determined in addition to SAP activities. At Q_o/V = 1 vvm air flow rate, the effect of agitation rate on DO concentration, pH, product, and by-product concentrations and SAP activity were investigated at N = 150, 500, and 750 min⁻¹; these are named as low-(LOT), medium-(MOT), and high oxygen transfer (HOT) conditions. LOT conditions favor biomass concentration; however, substrate consumption was highest at HOT conditions. MOT was optimum for maximum SAP activity which was 441 U cm⁻³ at t = 37 h. The total amino acid concentration was maximum in LOT and minimum in MOT conditions; lysine had the highest concentration under all oxygen transfer conditions. Among organic acids, acetic acid had the highest concentration and its concentration increased with oxygen transfer rate. The oxygen transfer coefficient increases with the agitation rate and the oxygen consumption rate increased almost linearly with the biomass concentration.     

Oxygen transfer kinetics of riboflavin fermentation by Ashbya gossypii in agitated fermentors

Effects of agitation and aeration rates on volumetric oxygen transfer coefficient and oxygen uptake rate of a riboflavin broth containing Ashbya gossypii were investigated in three batch, sparged, and agitated fermentors having the working volumes of 0.42, 0.85, and 2.5 l. The change of oxygen uptake rate with time at 250 rev min⁻¹ stirring and vvm aeration rates was shown. The volumetric oxygen transfer coefficients and maximum oxygen uptake rates obtained have been correlated to mechanical power inputs per unit volume of the fermentation broth and the superficial air velocities.     

动物乳腺生物反应器的现状和趋势

利用转基因家畜的乳腺生产人类重组蛋白,可以高效获得安全、足量的药用蛋白。本文针对乳腺生物反应器的成功研制,从目的基因的选择、载体构建、转基因技术等方面探讨了动物乳腺生物反应器的研究现状。分析了提高转基因效率和外源蛋白表达水平的技术途径,提出了降低总体成本的战略措施。特别探讨了利用Cre-loxP系统发展“体细胞打靶体细胞核移植技术体系”,高效生产乳腺生物反应器动物的可能性。     

动物乳腺生物反应器研究进展

利用转基因家畜的乳腺生产人类重组蛋白,可以高效获得安全、足量的药用蛋白。本在简要介绍乳腺生物反应器的基本原理及优越性的基础上,对其目的基因、表达载体和转基因技术在国内外的研究现状加以综述,着重探讨了体细胞核移植方法生产乳腺生物反应器的优越性及面临的技术问题。     

Verification of energy dissipation rate scalability in pilot and production scale bioreactors using computational fluid dynamics

Suspension mammalian cell cultures in aerated stirred tank bioreactors are widely used in the production of monoclonal antibodies. Given that production scale cell culture operations are typically performed in very large bioreactors (≥ 10,000 L), bioreactor scale‐down and scale‐up become crucial in the development of robust cell‐culture processes. For successful scale‐up and scale‐down of cell culture operations, it is important to understand the scale‐dependence of the distribution of the energy dissipation rates in a bioreactor. Computational fluid dynamics (CFD) simulations can provide an additional layer of depth to bioreactor scalability analysis. In this communication, we use CFD analyses of five bioreactor configurations to evaluate energy dissipation rates and Kolmogorov length scale distributions at various scales. The results show that hydrodynamic scalability is achievable as long as major design features (# of baffles, impellers) remain consistent across the scales. Finally, in all configurations, the mean Kolmogorov length scale is substantially higher than the average cell size, indicating that catastrophic cell damage due to mechanical agitation is highly unlikely at all scales. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:760–764, 2014