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     

Characterizing the fluid dynamics in the flow fields of cylindrical orbitally shaken bioreactors with different geometry sizes

Orbitally shaken bioreactors (OSRs) are commonly used for the cultivation of mammalian cells in suspension. To aid the geometry designing and optimizing of OSRs, we conducted a three‐dimensional computational fluid dynamics (CFD) simulation to characterize the flow fields in a 10 L cylindrical OSR with different vessel diameters. The liquid wave shape captured by a camera experimentally validated the CFD models established for the cylindrical OSR. The geometry size effect on volumetric mass transfer coefficient (k_La) and hydromechanical stress was analyzed by varying the ratio of vessel diameter (d) to liquid height at static (h_L), d/h_L. The highest value of k_La about 30 h^?1 was observed in the cylindrical vessel with the d/h_L of 6.35. Moreover, the magnitudes of shear stress and energy dissipation rate in all the vessels tested were below their minimum values causing cells damage separately, which indicated that the hydromechanical‐stress environment in OSRs is suitable for cells cultivation in suspension. Finally, the CFD results suggested that the d/h_L higher than 8.80 should not be adopted for the 10 L cylindrical OSR at the shaking speed of 180 rpm because the “out of phase” state probably will happen there.     

A micro-jet array for economic intensification of gas transfer in bioreactors

Bioreactors are of interest for gas-to-liquid conversion of stranded or waste industrial gases, such as CO, CH₄, or syngas. Process economics requires reduction of bioreactor cost and size while maintaining intense production via rapid delivery of gases to the liquid phase (i.e., high k_La). Here, we show a novel bioreactor design that outperforms all known technology in terms of gas transfer energy efficiency (k_La per power density) while operating at high k_La (i.e., near 0.8 s⁻¹). The reactor design uses a micro-jet array to break feedstock gas into a downward microbubble flow. Hydrodynamic and surfactant measurements show the reactor's advanced performance arises from its bubble breakage mechanism, which limits fluid shear to a thin plane located at an optimal location for bubble breakage. Power dissipation and k_L are shown to scale with micro-jet diameter rather than reactor diameter, and the micro-jet array achieves improved performance compared to classical impinging-jets, ejector, or U-loop reactors. The hydrodynamic mechanism by which the micro-jets break bubbles apart is shown to be shearing the bubbles into filaments then fragmentation by surface tension rather than “cutting in half” of bubbles. Guided by these hydrodynamic insights, strategies for industrial design are given. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2710, 2019     

A test facility for fritted spargers of production-scale-bioreactors

The production of therapeutic proteins requires qualification of equipment components and appropriate validation procedures for all operations. Since protein productions are typically performed in bioreactors using aerobic cultivation processes air sparging is an essential factor. As recorded in literature, besides ring spargers and open pipe, sinter frits are often used as sparging elements in large scale bioreactors. Due to the manufacturing process these frits have a high lot-to-lot product variability. Experience shows this is a practical problem for use in production processes of therapeutic proteins, hence frits must be tested before they can be employed. The circumstance of checking quality and performance of frits as sparging elements was investigated and various possibilities have been compared. Criteria have been developed in order to evaluate the sparging performance under conditions comparable to those in production bioreactors. The oxygen mass transfer coefficient (k _L a) was chosen as the evaluation criterion. It is well known as an essential performance measure for fermenters in the monoclonal antibody production. Therefore a test rig was constructed able to automatically test frit-spargers with respect to their k _L a-values at various gas throughputs. Performance differences in the percent range could be detected.     

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     

Characterizing the fluid dynamics of the inverted frustoconical shaking bioreactor

The authors conducted a three‐dimensional computational fluid dynamics (CFD) simulation to calculate the flow field in the inverted frustoconical shaking bioreactor with 5 L working volume (IFSB‐5L). The CFD models were established for the IFSB‐5L at different operating conditions (different shaking speeds and filling volumes) and validated by comparison of the liquid height distribution in the agitated IFSB‐5L. The “out of phase” operating conditions were characterized by analyzing the flow field in the IFSB‐5L at different filling volumes and shaking speeds. The values of volumetric power consumption (P/V_L) and volumetric mass transfer coefficient (k_La) were determined from simulated and experimental results, respectively. Finally, the operating condition effect on P/V_L and k_La was investigated. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:478–485, 2018     

Enhancement of oxygen mass transfer in stirred bioreactors using oxygen-vectors. 1. Simulated fermentation broths

Oxygen mass transfer represents the most important parameter involved in the design and operation of mixing-sparging equipment for bioreactors. It can be described and analyzed by means of the mass transfer coefficient, k_La. The k_La values are affected by many factors such as geometrical and operational characteristics of the vessels, media composition, type, concentration and microorganism morphology, and biocatalysts properties. The efficiency of oxygen transfer could be enhanced by adding oxygen-vectors in broths, such as hydrocarbons or fluorocarbons, without increasing the energy consumption for mixing or aeration. The experimental results obtained for simulated broths indicated a considerable increase of k_La in the presence of n-dodecane, and the existence of a certain value of n-dodecane concentration that corresponds to a maximum mass transfer rate of oxygen. The magnitude of the positive effect of n-dodecane depends both on the broths characteristics and operational conditions of the bioreactor.Notation d stirrer diameter, mm - d oxygen electrode diameter, mm - D bioreactor diameter, mm - h distance from the inferior stirrer to the bioreactor bottom, mm - H bioreactor height, mm - k_La oxygen mass transfer coefficient, s^-1 - l impeller blade length, mm - I oxygen electrode immersed length, mm - P power consumption for mixing of non-aerated broths, W - P_a power consumption for mixing of aerated broths, W - (P_a/V) specific power input, W/m³ - s baffle width, mm - v_S superficial air velocity, m/s - V volume of medium, m³ - w impeller blade height, mm - volumetric fraction of oxygen-vector - _a apparent viscosity, Pa*s - density, kg/m³     

Effect of the impeller-sparger configuration over Trichoderma harzianum growth in four-phases cultures under constant dissolved oxygen

Three impeller-sparger configurations were used to evaluate the effect of different hydrodynamic conditions over fungal growth in rheologically complex cultures of Trichoderma harzianum using castor oil as sole carbon source. Three spargers (ring, sintered and 5-orifice) in combination with a turbine impeller system "TIS" (two Rushton turbines) or a hybrid impeller system "HIS" (Rushton turbine and a marine propeller as lower and upper impellers) were used. Their performance was assessed in terms of the response towards disturbance (PID oxygen control settings) and oxygen mass transfer (k_La). To avoid oxygen limitations, all cultures were controlled at 10% DOT by gas blending. Top to bottom mixing, and hence bulk blending, was improved when the - axial flow - HIS was used, ensuring phase interaction and substrate (oil) circulation. The 5-orifice sparger in combination with the TIS configuration yielded the longest lag phase and lowest k_La due to poor bulk blending and to the low gas-liquid interfacial area developed. The highest k_La was achieved with the sintered sparger-HIS probably due to considerable interfacial bubble area enhancement. However, growth limitation occurred as consequence of poor substrate availability as a stable air-oil emulsion was formed at the top of the tank. The best compromise between bulk blending (phase interaction), oxygen transfer (k_La) and fungal growth (growth rate) was achieved with the ring sparger-HIS configuration.     

Comparison of the hydrodynamics and mass transfer characteristics in internal-loop airlift bioreactors utilizing either a novel membrane-tube sparger or perforated plate sparger

Two gas spargers, a novel membrane-tube sparger and a perforated plate sparger, were compared in terms of hydrodynamics and mass transfer (or oxygen transfer) performance in an internal-loop airlift bioreactor. The overall gas holdup ε _T, downcomer liquid velocity V _d, and volumetric mass transfer coefficient K _L a were examined depending on superficial gas velocity U _G increased in Newtonian and non-Newtonian fluids for the both spargers. Compared with the perforated plate sparger, the bioreactor with the membrane-tube sparger increased the values of ε _T by 4.9–48.8 % in air–water system when the U _G was from 0.004 to 0.04 m/s, and by 65.1–512.6 % in air–CMC solution system. The V _d value for the membrane-tube sparger was improved by 40.0–86.3 %. The value of K _L a was increased by 52.8–84.4 % in air–water system, and by 63.3–836.3 % in air–CMC solution system. Empirical correlations of ε _T, V _d, and K _L a were proposed, and well corresponding with the experimental data with the deviation of 10 %.     

Hydrodynamics and mass transfer in miniaturized bubble column bioreactors

Miniaturized bubble columns (MBCs) have different hydrodynamics in comparison with the larger ones, but there is a lack of scientific data on MBCs. Hence, in this study, the effect of gas hold-up, flow regimes, bubble size distribution on volumetric oxygen mass transfer coefficient at different pore size spargers and gas flow rates in MBCs in the presence and absence of microorganisms were investigated. It was found that flow regime transition occurred around low gas flow rates of 1.18 and 0.85 cm/s for small (16–40 µm) and large (40–100 µm) pore size spargers, respectively. Gas hold-up and K_La in MBC with small size sparger were higher than those with larger one, with an increasing effect in the presence of microorganisms. A comparison revealed that the wall effect on the flow regime and gas hold-up in MBCs was greater than bench-scale bubble columns. The K_La values significantly increased up to tenfold using small pore size sparger. In the MBC and stirred tank bioreactors, the maximum obtained cell concentrations were OD₆₀₀ of 41.5 and 43.0, respectively. Furthermore, it was shown that in MBCs, higher K_La and lower turbulency could be achieved at the end of bubbly flow regime.

     

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.     

Production and partial characterization of biosurfactant produced by Streptomyces sp. R1

The present study focused on developing a wild-type actinomycete isolate as a model for a non-pathogenic filamentous producer of biosurfactants. A total of 33 actinomycetes isolates were screened and their extracellular biosurfactants production was evaluated using olive oil as the main substrate. Out of 33 isolates, 32 showed positive results in the oil spreading technique (OST). All isolates showed good emulsification activity (E₂₄) ranging from 84.1 to 95.8%. Based on OST and E₂₄ values, isolate R1 was selected for further investigation in biosurfactant production in an agitated submerged fermentation. Phenotypic and genotypic analyses tentatively identified isolate R1 as a member of the Streptomyces genus. A submerged cultivation of Streptomyces sp. R1 was carried out in a 3-L stirred-tank bioreactor. The influence of impeller tip speed on volumetric oxygen transfer coefficient (k _L a), growth, cell morphology and biosurfactant production was observed. It was found that the maximum biosurfactant production, indicated by the lowest surface tension measurement (40.5 ± 0.05 dynes/cm) was obtained at highest k _L a value (50.94 h⁻¹) regardless of agitation speed. The partially purified biosurfactant was obtained at a concentration of 7.19 g L⁻¹, characterized as a lipopeptide biosurfactant and was found to be stable over a wide range of temperature (20–121 °C), pH (2–12) and salinity [5–20% (w/v) of NaCl].

     

Understanding the effect of high gas entrance velocity on Chinese hamster ovary (CHO) cell culture performance and its implications on bioreactor scale-up and sparger design

There are three main potential sources for cell shear damage existing in stirred tank bioreactors. One is the potential high energy dissipation in the immediate impeller zones; another from small gas bubble burst; and third is from high gas entrance velocity (GEV) emitting from the sparger. While the first two have been thoroughly addressed for the scale-up of Chinese hamster ovary (CHO) cell culture knowing that a wide tolerable agitation range with non-damaging energy dissipation exists and the use of shear protectants like Pluronic F68 guard against cell damage caused by bubble burst, GEV remains a potential scale-up problem across scales for the drilled hole or open pipe sparger designs. GEV as high as 170 m/s due to high gas flow rates and relatively small sparger hole diameters was observed to be significantly detrimental to cell culture performance in a 12,000 L bioreactor when compared to a satellite 2 L bioreactor run with GEV of <1 m/s. Small scale study of GEV as high as 265 m/s confirmed this. Based on the results of this study, a critical GEV of >60 m/s for CHO cells is proposed, whereas previously 30 m/s has been reported for NS0 cells by Zhu, Cuenca, Zhou, and Varma (2008. Biotechnol. Bioeng., 101, 751–760). Implementation of new large scale spargers with larger diameter and more holes lowered GEV and helped improve the cell culture performance, closing the scale-up gap. Design of such new spargers was even more critical when hole plugging was discovered during large scale cultivation hence exacerbating the GEV impact. Furthermore, development of a scale down model based on mimicry of the large scale GEV profile as a function of time was proven to be beneficial for reproducing large scale results.     

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.     

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     

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.     

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.     

Improvement of foam breaking and oxygen-transfer performance in a stirred-tank fermenter

This study examined a stirred-tank fermenter (STF) containing low-viscosity foaming liquids with an agitation impeller and foam-breaking impeller mounted on the same shaft. Results showed that the performance of the foam-breaking impeller can be improved by changing a conventional six-blade turbine impeller into a rod impeller as the agitation impeller. The volumetric oxygen-transfer coefficient, k _L a, in the mechanical foam-control method (MFM) using a six-blade vaned disk as the foam-breaking impeller in the STF with the rod impeller was approximately five times greater than that of the chemical foam-control method (CFM) adding an anti-foaming agent in the STF with the six-blade turbine impeller. Application of the present method to the cultivation of Saccharomyces cerevisiae K-7 demonstrated that the cultivation time up to the maximum cell concentration was remarkably shorter than that achieved using a conventional CFM.     

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.