Using CFD simulations and statistical analysis to correlate oxygen mass transfer coefficient to both geometrical parameters and operating conditions in a stirred-tank bioreactor

Optimization of a bioreactor design can be an especially challenging process. For instance, testing different bioreactor vessel geometries and different impeller and sparger types, locations, and dimensions can lead to an exceedingly large number of configurations and necessary experiments. Computational fluid dynamics (CFD), therefore, has been widely used to model multiphase flow in stirred-tank bioreactors to minimize the number of optimization experiments. In this study, a multiphase CFD model with population balance equations are used to model gas–liquid mixing, as well as gas bubble distribution, in a 50 L single-use bioreactor vessel. The vessel is the larger chamber in an early prototype of a multichamber bioreactor for mammalian cell culture. The model results are validated with oxygen mass transfer coefficient (k_La) measurements within the prototype. The validated model is projected to predict the effect of using ring or pipe spargers of different sizes and the effect of varying the impeller diameter on k_La. The simulations show that ring spargers result in a superior k_La compared to pipe spargers, with an optimum sparger-to-impeller diameter ratio of 0.8. In addition, larger impellers are shown to improve k_La. A correlation of k_La is presented as a function of both the reactor geometry (i.e., sparger-to-impeller diameter ratio and impeller-to-vessel diameter ratio) and operating conditions (i.e., Reynolds number and gas flow rate). The resulting correlation can be used to predict k_La in a bioreactor and to optimize its design, geometry, and operating conditions.     

Syngas fermentation to biofuel: Evaluation of carbon monoxide mass transfer coefficient (kLa) in different reactor configurations

Lignocellulosic biomass such as agri‐residues, agri‐processing by‐products, and energy crops do not compete with food and feed, and is considered to be the ideal renewable feedstocks for biofuel production. Gasification of biomass produces synthesis gas (syngas), a mixture primarily consisting of CO and H₂. The produced syngas can be converted to ethanol by anaerobic microbial catalysts especially acetogenic bacteria such as various clostridia species.One of the major drawbacks associated with syngas fermentation is the mass transfer limitation of these sparingly soluble gases in the aqueous phase. One way of addressing this issue is the improvement in reactor design to achieve a higher volumetric mass transfer coefficient (k_La). In this study, different reactor configurations such as a column diffuser, a 20‐μm bulb diffuser, gas sparger, gas sparger with mechanical mixing, air‐lift reactor combined with a 20‐μm bulb diffuser, air‐lift reactor combined with a single gas entry point, and a submerged composite hollow fiber membrane (CHFM) module were employed to examine the k_La values. The k_La values reported in this study ranged from 0.4 to 91.08 h^?1. The highest k_La of 91.08 h^?1 was obtained in the air‐lift reactor combined with a 20‐μm bulb diffuser, whereas the reactor with the CHFM showed the lowest k_La of 0.4 h^?1. By considering both the k_La value and the statistical significance of each configuration, the air‐lift reactor combined with a 20‐μm bulb diffuser was found to be the ideal reactor configuration for carbon monoxide mass transfer in an aqueous phase. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools

During the past decade, novel disposable cell culture vessels (generally referred to as Process Scouting Devices or PSDs) have become increasingly popular for laboratory scale studies and seed culture generation. However, the lack of engineering characterization and online monitoring tools for PSDs makes it difficult to elucidate their oxygen transfer capabilities. In this study, a mass transfer characterization (k_La) of sensor enabled static and rocking T‐flasks is presented and compared with other non‐instrumented PSDs such as CultiFlask 50®, spinner flasks, and SuperSpinner D 1000®. We have also developed a mass transfer empirical correlation that accounts for the contribution of convection and diffusion to the volumetric mass transfer coefficient (k_La) in rocking T‐flasks. We also carried out a scale‐down study at matched k_La between a rocking T75‐flask and a 10 L (2 L filling volume) wave bioreactor (Cultibag®) and we observed similar DO and pH profiles as well as maximum cell density and protein titer. However, in this scale‐down study, we also observed a negative correlation between cell growth and protein productivity between the rocking T‐flask and the wave bioreactor. We hypothesize that this negative correlation can be due to hydrodynamic stress difference between the rocking T‐flask and the Cultibag. As both cell culture devices share key similarities such as type of agitation (i.e., rocking), oxygen transfer capabilities (i.e., k_La) and disposability, we argue that rocking T‐flasks can be readily integrated with wave bioreactors, making the transition from research‐scale to manufacturing‐scale a seamless process. Biotechnol. Bioeng. 2012;109: 2295–2305. © 2012 Wiley Periodicals, Inc.     

A new dynamic model for highly efficient mass transfer in aerated bioreactors and consequences for kLa identification

Gas–liquid mass transfer is often rate‐limiting in laboratory and industrial cultures of aerobic or autotrophic organisms. The volumetric mass transfer coefficient k_La is a crucial characteristic for comparing, optimizing, and upscaling mass transfer efficiency of bioreactors. Reliable dynamic models and resulting methods for parameter identification are needed for quantitative modeling of microbial growth dynamics. We describe a laboratory‐scale stirred tank reactor (STR) with a highly efficient aeration system (k_La ≈ 570 h^?1). The reactor can sustain yeast culture with high cell density and high oxygen uptake rate, leading to a significant drop in gas concentration from inflow to outflow (by 21%). Standard models fail to predict the observed mass transfer dynamics and to identify k_La correctly. In order to capture the concentration gradient in the gas phase, we refine a standard ordinary differential equation (ODE) model and obtain a system of partial integro‐differential equations (PIDE), for which we derive an approximate analytical solution. Specific reactor configurations, in particular a relatively short bubble residence time, allow a quasi steady‐state approximation of the PIDE system by a simpler ODE model which still accounts for the concentration gradient. Moreover, we perform an appropriate scaling of all variables and parameters. In particular, we introduce the dimensionless overall efficiency κ, which is more informative than k_La since it combines the effects of gas inflow, exchange, and solution. Current standard models of mass transfer in laboratory‐scale aerated STRs neglect the gradient in the gas concentration, which arises from highly efficient bubbling systems and high cellular exchange rates. The resulting error in the identification of κ (and hence k_La) increases dramatically with increasing mass transfer efficiency. Notably, the error differs between cell‐free and culture‐based methods of parameter identification, potentially confounding the determination of the “biological enhancement” of mass transfer. Our new model provides an improved theoretical framework that can be readily applied to aerated bioreactors in research and biotechnology. Biotechnol. Bioeng. 2012; 109: 2997–3006. © 2012 Wiley Periodicals, Inc.     

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the k_La increased with the gas fractional hold-up (ε_G) and the aeration rate. A linear relationship between ε_G and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher k_La and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets.

Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns.

Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.     

Enhancement of anthocyanin production by Perilla frutescens cells in a stirred bioreactor with internal light irradiation

Oxygen supply and light irradiation exhibited significant influence on the production of anthocyanin (red pigments) by suspended cultures of Perilla frutescens cells in a 2.6-l aerated and agitated bioreactor with a six-flat-bladed turbine. When the initial volumetric oxygen transfer coefficient (k_La) value was below 10 h⁻¹ and light was not irradiated, the anthocyanin production was never over 0.6 g/l. By modification of a gas sparger, the oxygen supply capability of the bioreactor was remarkably improved, and 1.65 g/l of anthocyanin was obtained at an enhanced k_La value of 15.4 h⁻¹. Moreover, it was found that anthocyanin accumulation at a 0.2 vvm aeration rate was higher than that at 0.1 or 0.4 vvm in the modified bioreactor, with the other cultivation conditions kept the same. Light irradiation also significantly increased anthocyanin accumulation in the stirred reactor at a low k_La value, i.e. 9.9 h⁻¹. However, a combination of irradiation with a higher oxygen supply reduced the production of anthocyanin in the bioreactor.     

A comparative evaluation of oxygen mass transfer and broth viscosity using Cephalosporin-C production as a case strategy

The production of Cephalosporin-C (CPC) a secondary metabolite, using a mold Acremonium chrysogenum was studied in a lab scale Internal loop air lift reactor. Cephalosporin-C production process is a highly aerobic fermentation process. Volumetric gas–liquid mass transfer coefficient (k_La) and viscosity (η) were evaluated, during the growth and production phases of the microbial physiology. An attempt has been made to correlate the broth viscosity, η and volumetric oxygen transfer coefficient, k_La during the Cephalosporin-C production in an air lift reactor. The impact of biomass concentration and mycelial morphology on broth viscosity has been also evaluated. The broth exhibits a typical non-Newtonian fermentation broth. Rheology parameters like consistency index and fluidity index are also studied.     

Oxygen transfer characteristics of miniaturized bioreactor systems

Since their introduction in 2001 miniaturized bioreactor systems have made great advances in function and performance. In this article the dissolved oxygen (DO) transfer performance of submilliliter microbioreactors, and 1–10 mL minibioreactors was examined. Microbioreactors have reached k_La values of 460 h^?1, and are offering instrumentation and some functionality comparable to production systems, but at high throughput screening volumes. Minibioreactors, aside from one 1,440 h^?1 k_La system, have not offered as high rates of DO transfer, but have demonstrated superior integration with automated fluid handling systems. Microbioreactors have been typically limited to studies with E. coli, while minibioreactors have offered greater versatility in this regard. Further, mathematical relationships confirming the applicability of k_La measurements across all scales have been derived, and alternatives to fluorescence lifetime DO sensors have been evaluated. Finally, the influence on reactor performance of oxygen uptake rate (OUR), and the possibility of its real‐time measurement have been explored. Biotechnol. Bioeng. 2013; 110: 1005–1019. © 2012 Wiley Periodicals, Inc.     

Enhancement of mass transfer characteristics and phenanthrene degradation in a two-phase partitioning bioreactor equipped with internal static mixers

Oxygen and substrate supply have always been considered physical constraints for the performance and operation of two-phase partitioning bioreactors (TPPB), widely used for the degradation of hydrophobic substrates. In this regard, the potential advantages of static mixers in upgrading the oxygen transfer and liquid-liquid dispersions in TPPB have been highlighted. In the present paper, the concomitant influence of static mixers on the gas-liquid mass transfer coefficient k _L a and on substrate bioavailability was examined in TPPB. The static method based on conventional forms was developed to estimate the oxygen volumetric mass transfer coefficient. Over a broad range of liquid and air flow rates, the presence of static mixers was found to significantly enhance k _L a relative to a mixer-free mode of operation. For identical conditions, static mixers improved the k _L a threefold. In the presence of external aeration supply, the boost in the k _L a was associated with an increase of 16% in the phenanthrene biodegradation rate due to bubble break up accomplished by the static mixers. On the other hand, static mixers were efficient in enhancing substrate bioavailability by improving the liquid-liquid interfacial area. This effect was reflected by a threefold increase in the degradation rate in the bioreactors with no external supply of air when equipped with static mixers.     

Submerged monoxenic culture of the entomopathogenic nematode, <Emphasis Type="Italic">Steinernema carpocapsae</Emphasis> CABA01, in a mechanically agitated bioreactor: Evolution of the hydrodynamic and mass transfer conditions

This study is the first to describe the evolution of both hydrodynamic and oxygen transfer conditions during the submerged culture of the entomopathogenic nematode, Steinernema carpocapsae CABA01 (an indigenous strain isolated within the State of Hidalgo, Mexico), and its symbiotic bacterium, Xenorhabdus nematophila, using an internal-loop mechanically agitated bioreactor of 4.5 L of liquid volume. Concentrations up to 217,306 viable nematodes per mL, with 94% in infective juvenile (IJ) stage (i.e., 204,444 IJ/mL), were achieved in 16 days of bioprocess. The Reynolds number (Re) was used as an index of the actual hydrodynamic conditions, and it varied within the interval 5,150 < Re (dimensionless) < 9,440, involving apparent culture broth viscosity changes from 3 to 5.4 mPa s during the processing. The aeration efficiency was expressed on the basis of the volumetric oxygen transfer coefficient, k _L a, which varied within the range 0.026 to 0.170 s⁻¹.     

Oxygen mass transfer and scale-up studies in baffled roller bioreactors

Oxygen mass transfer was studied in conventional, bead mill and baffled roller bioreactors. Using central composite rotational design, impacts of size, rotation speed and working volume on the oxygen mass transfer were evaluated. Baffled roller bioreactor outperformed its conventional and bead mill counterparts, with the highest k _L a obtained in these configurations being 0.58, 0.19, 0.41 min^?1, respectively. Performances of the bead mill and baffled roller bioreactor were only comparable when a high bead loading (40 %) was applied. Regardless of configuration increase in rotation speed and decrease in working volume improved the oxygen mass transfer rate. Increase in size led to enhanced mass transfer and higher k _L a in baffled roller bioreactor (0.49 min^?1 for 2.2 L and 1.31 min^?1 for 55 L bioreactors). Finally, the experimentally determined k _L a in the baffled roller bioreactors of different sizes fit reasonably well to an empirical correlation describing the k _L a in terms of dimensionless numbers.     

Oxygen mass-transfer performance of low viscosity gas-liquid-solid system in a split-cylinder airlift bioreactor

The O₂ mass-transfer coefficient, k _L a, decreased by 20% when the viscosity of a simulated broth increased from 1.38 × 10^–3 to 3.43 × 10^–3 Pa s in a split-cylinder airlift bioreactor with a broth volume of 41 l. When the paper pulp concentration was below 10 g l^–1, k _L a hardly changed. While at 30 g l^–1, k _L a decreased by 56%. C₂O₄ ^2– and Na⁺ were found to have some effect on the k _L a value.     

CFD of mixing of multi‐phase flow in a bioreactor using population balance model

Mixing in bioreactors is known to be crucial for achieving efficient mass and heat transfer, both of which thereby impact not only growth of cells but also product quality. In a typical bioreactor, the rate of transport of oxygen from air is the limiting factor. While higher impeller speeds can enhance mixing, they can also cause severe cell damage. Hence, it is crucial to understand the hydrodynamics in a bioreactor to achieve optimal performance. This article presents a novel approach involving use of computational fluid dynamics (CFD) to model the hydrodynamics of an aerated stirred bioreactor for production of a monoclonal antibody therapeutic via mammalian cell culture. This is achieved by estimating the volume averaged mass transfer coefficient (k_La) under varying conditions of the process parameters. The process parameters that have been examined include the impeller rotational speed and the flow rate of the incoming gas through the sparger inlet. To undermine the two‐phase flow and turbulence, an Eulerian‐Eulerian multiphase model and k‐ε turbulence model have been used, respectively. These have further been coupled with population balance model to incorporate the various interphase interactions that lead to coalescence and breakage of bubbles. We have successfully demonstrated the utility of CFD as a tool to predict size distribution of bubbles as a function of process parameters and an efficient approach for obtaining optimized mixing conditions in the reactor. The proposed approach is significantly time and resource efficient when compared to the hit and trial, all experimental approach that is presently used. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:613–628, 2016     

Substrate effects in tower loop reactors

Summary Hansenula polymorpha was cultivated in a bubble column loop bioreactor employing ethanol and/or glucose as substrates. By varying the substrate concentration, the cultivations were carried out in non-limited, substrate limited and oxygen transfer limited growth ranges. The influence of the transitions from one range to another on reactor performance (OTR,k _L a, a) and cell productivity () were investigated. When employing ethanol as a substrate, the concentration considerably influences the fluid dynamics, mass transfer phenomena and cell productivity. When employing glucose as a substrate, glucose repression occurs. At high glucose concentrations no transition into the oxygen transfer limited growth is possible. The ethanol produced during the glucose repression influences the fluid dynamics, mass transfer phenomena and productivity. With decreasing glucose concentration the glucose repression can be gradually eliminated.     

The influence of polymeric membrane gas spargers on hydrodynamics and mass transfer in bubble column bioreactors

Gas sparging performances of a flat sheet and tubular polymeric membranes were investigated in 3.1 m bubble column bioreactor operated in a semi batch mode. Air–water and air–CMC (Carboxymethyl cellulose) solutions of 0.5, 0.75 and 1.0 % w/w were used as interacting gas–liquid mediums. CMC solutions were employed in the study to simulate rheological properties of bioreactor broth. Gas holdup, bubble size distribution, interfacial area and gas–liquid mass transfer were studied in the homogeneous bubbly flow hydrodynamic regime with superficial gas velocity (U _G) range of 0.0004–0.0025 m/s. The study indicated that the tubular membrane sparger produced the highest gas holdup and densely populated fine bubbles with narrow size distribution. An increase in liquid viscosity promoted a shift in bubble size distribution to large stable bubbles and smaller specific interfacial area. The tubular membrane sparger achieved greater interfacial area and an enhanced overall mass transfer coefficient (K _La) by a factor of 1.2–1.9 compared to the flat sheet membrane.     

Effects of hydrocarbon additions on gas-liquid mass transfer coefficients in biphasic bioreactors

The effects of aliphatic hydrocarbons (n-hexadecane andn-dodecane) on the volumetric oxygen mass transfer coefficient (k _L a) were studied in flat alveolar airlift reactor and continuous stirred tank reactors (CSTRs). In the flat alveolar airlift reactor, high aeration rates (>2 vvm) were required in order to obtain efficient organic-aqueous phase dispersion and reliablek _L a measurements. Addition of 1% (v/v)n-hexadecane orn-dodecane increased thek _l a 1.55-and 1.33-fold, respectively, compared to the control (superficial velocity: 25.8×10⁻³ m/s, sparger orifice diameter: 0.5 mm). Analysis of the gas-liquid interfacial areaa and the liquid film mass transfer coefficientk _L suggests that the observedk _L a increase was a function of the media's liquid film mass transfer. Addition of 1% (v/v)n-hexadecane orn-dodecane to analogous setups using CSTRs led to ak _L a increase by a factor of 1.68 and 1.36, respectively (superficial velocity: 2.1×10⁻³ m/s, stirring rate: 250 rpm). These results propose that low-concentration addition of oxygen-vectors to aerobic microbial cultures has additional benefit relative to incubation in purely aqueous media.     

Oxygen mass transfer and gas holdup in a bubble column bioreactor with biosynthesis liquids

The influence of the rheology of some antibiotic biosynthesis liquids produced by Streptomyces aureofaciens, Nocardia mediterranei and Penicillium chrysogenum on the volumetric liquid phase oxygen transfer coefficient, k_La, and gas holdup, ε_G, together with the influence of superficial gas velocity, were studied in a bubble column bioreactor, using samples of fermentation liquids taken from industrial stirred tank fermenters, at 30-hour intervals during fermentation batch. The results were compared to those of previous studies from literature on non-Newtonian homogeneous fluids, such as CMC-Na, xanthan and starch solutions, respectively. In the heterogeneous broths, ε_G and k_La decreased with increasing apparent viscosity of the broth and increased with increasing superficial velocity. The experimental data were correlated using non-linear regression with correlation coefficients above 0.85.     

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     

Performance of an external loop air-lift bioreactor for the production of monoclonal antibodies by immobilized hybridoma cells

Summary The performance of an external loop air-lift bioreactor was investigated by assessing the inter-relationships between various hydrodynamic properties and mass transfer. The feasibility of using this bioreactor for the production of monoclonal antibodies by mouse hybridoma cells immobilized in calcium alginate gel beads and alginate/poly-l-lysine microcapsules was also examined. When the superficial gas velocity, V _g , in the 300 ml reactor was varied from 2 to 36 cm/min, the average liquid velocity increased from 3 to 14 cm/sec, the gas hold-up rose from 0.2 to 3.0%, and the oxygen mass transfer coefficient, k _L a, increased from 2.5 to 18.1 h^-1. A minimum liquid velocity of 4 cm/s was required to maintain alginate gel beads (1000 m diameter, occupying 3% of reactor volume) in suspension. Batch culture of hybridoma cells immobilized in alginate beads followed logarithmic growth, reaching a concentration of 4×10⁷ cells/ml beads after 11 days. Significant antibody production did not occur until day 9 into the culture, reaching a value of 100 g/ml of medium at day 11. On the other hand, bioreactor studies with encapsulated hybridoma cells gave monoclonal antibody concentrations of up to 800 g/ml capsules (the antibody being retained within the semipermeable capsule) and maximum cell densities of 2×10⁸ cells/ml capsule at day 11. The volumetric productivities of the alginate gel immobilized cell system and the encapsulated cell system were 9 and 3 g antibody per ml of reactor volume per day, respectively. The main advantage of the bioreactor system is its simple design, since no mechanical input is required to vary the hydrodynamic properties.