Membrane sparger in bubble column,airlift, and combined membrane–ring sparger bioreactors

The bubble column and the two internal loop airlift reactors (riser/downcomer area ratios of 0.11 and 0.58) characterized in this study were equipped with a rubber membrane sparger, which produced small bubbles, giving high mass transfer coefficients. The low mixing intensity in the bubble column was increased by an order of magnitude in the airlift reactors. We designed a novel aeration and mixing system by adding a ring sparger to the membrane sparger in the bubble column and maintained the advantages of both airlift configuration (good mixing properties) and bubble column configuration (efficient aeration, without any internal constructions). The combined membrane–ring sparger system has unique features with respect to the efficiency of utilization of substrate gasses and energy. Model experiments showed that the small bubbles from the membrane sparger do not coalesce with the large bubbles from the ring sparger. If different gases were added through the two spargers it was possible to transfer a hazardous or expensive gas quantitatively to the liquid through the membrane sparger (dual sparging mode). In the combined membrane–ring sparger system the energy input for mixing and mass transfer is divided. Therefore, the energy consumption can be minimized if the flow distribution of air through the membrane and ring sparger is controlled by the oxygen demand and the inhomogeneity of the culture, respectively (split sparging mode). The dual sparging mode was used for mass production of the alga Rhodomonas sp. as the first step in aquatic food chains. Avoiding mechanical parts removes an important risk of malfunction, and a continuous culture could be maintained for more than 8 months. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 452–458, 1999.     

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.     

Experimental study of a ceramic microsparging aeration system in a pilot-scale animal cell culture

The oxygen supply of cell cultures with the aid of free gas bubbles is an efficient process strategy in pharmaceutical production. If the cell-damaging impact of gas bubbles is reduced, direct aeration becomes a practical solution with scale-up potential and comparatively high oxygen transfer rates. In this paper a microsparging aeration system made of porous ceramic was compared with bubble-free membrane aeration. The sparging system was used for the long-term cultivation of mammalian cells in 2- to 100-L scale bioreactors and produced bubble sizes of 100-500 microm in diameter. Using a scale of 2.5 and 30 L, a cell density of 2.6 x 10(6) cells/mL was attained. When a 100-L scale was used, a density of 1.1 x 10(6) cells/mL was achieved, whereas a comparable membrane-aerated system showed a cell density of 2.2 x 10(6) cells/mL. At relatively low agitation rates of less than 70 rpm in the sparged bioreactors, a homogeneous and constant oxygen concentration was kept in the medium. As a result of the different foam-forming tendency caused by the lower gas flow of the ceramic sparger compared to that of the standard aeration systems, we were able to develop an appropriate process control strategy. Furthermore, oxygen transfer measurements for the common stainless steel sparger and the ceramic sparger showed a 3-fold higher oxygen transfer coefficient for the ceramic sparger.     

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     

An economical 20 litre bench-top fermenter

We describe an economical 20 litre bench-top fermenter suitable for production of recombinant antibody fragments in bacterial expression systems. The bacterial culture contained within a polycarbonate carboy is mixed (400-600 rpm) and aerated (1 vessel vol./min) by a high-shear radial flow impeller mounted on a hollow stainless steel shaft, through which pressurised air is pumped. Air is dispersed as fine bubbles into the culture medium by the turbine impeller, without the need for a porous sparger. A stainless steel baffle stabilised by a gliding counterweight increases mixing. The components can easily be disassembled for cleaning and sterilisation. Temperature (range 20-37 degrees C) and pH (range 7.0-7.5) are controlled manually. Using the apparatus, it proved possible to achieve Escherichia coli cell culture densities equivalent to an optical density at 600 nm (OD(600)) of 30-32, compared with OD(600) 4-6 in shake flasks. A yield of 40 mg/litre/day of a recombinant antibody fragment was obtained with the fermenter, which was 15-fold more than the yield of 2.5mg/litre/day achieved in shake flasks. The fermenter may be particularly suited for research purposes.     

Process-engineering characterization of small-scale bubble columns for microbial process development

Volumetric oxygen transfer rates and power inputs were estimated by a model of the formation of primary gas bubbles at the static sparger (sinter plate) of small-scale bubble columns and a common mass-transfer correlation for bubbles rising in a non-coalescent Newtonian electrolyte solution of low viscosity. Estimations were used to assess the dimensioning and possibilities of small-scale bubble column application with an height/diameter ratio of about 1. Estimations of volumetric oxygen transfer rates (<0.16 s^-1) and power inputs (<100 W m^-3) with a mean pore diameter of the static sparger of 13 µm were confirmed as function of the superficial air velocity (<0.6 cm s^-1) by measurements using an Escherichia coli fermentation medium. Small-scale bubble columns are thus to be classified between shaking flasks and stirred-tank reactors with respect to the oxygen transfer rate, but the maximum volumetric power input is more than one magnitude below the power input in shaking flasks, which is of the same order of magnitude as in stirred-tank reactors. A small-scale bubble columns system was developed for microbial process development, which is characterized by handling in analogy to shaking flasks, high oxygen transfer rates and simultaneous operation of up to 16 small-scale reactors with individual gas supply in an incubation chamber.     

Continuous production of citric acid with recirculation of the fermentation broth after product recovery

An airlift reactor with double net draft tubes was developed. A sparger was located between the two draft tubes. The draft tubes had a significant effect on breaking bubbles into smaller ones. The assessment of the reactor performance was based on gas holdup, mixing time, and volumetric mass transfer coefficient. The proposed reactor had higher gas holdup and volumetric mass transfer coefficient, and lower mixing time in comparison with those of the bubble column. Application of the proposed reactor to fermentation of Saccharomyces cerevisiae demonstrated that the cultivation time was significantly shortened.     

Effect of ventilation with soluble and diffusible gases on the size of air emboli

Pulmonary hypertension resulting from venous air embolism is known to increase after ventilation with highly soluble and diffusible gases. Exacerbation of the hypertension could be due to further blockage of the circulation if the bubbles enlarge as a result of ingress of gas by diffusion. This mechanism has been frequently cited but lacks direct proof. To determine directly whether intravascular air bubbles actually enlarge when highly soluble and diffusible gases are inspired, we used microscopy to measure the size of gas emboli in vivo. When air bubbles were injected into the right atrium, the bubbles that appeared in pulmonary arterioles were larger during ventilation with helium or nitrous oxide than with air. Air bubbles injected into the pulmonary artery enlarged when the inspired gas was changed to helium or nitrous oxide. The direction, magnitude, and timing of changes in bubble size were consistent with a net diffusion of gas into the bubbles. These data support the idea that venous air emboli enlarge during ventilation with soluble and diffusible gases and thereby cause further vascular obstruction.     

Response of <Emphasis Type="Italic">Tisochrysis lutea</Emphasis> [Prymnesiophycidae] to aeration conditions in a bench-scale photobioreactor

The effects of superficial gas velocity (U_gr), gas entrance velocity (ν), and bubble size on the growth of Tisochrysis lutea was investigated in 600-mL photobioreactors operated with airlift pumps. Superficial gas velocities, calculated from measured air flow rates, ranging from 7 to 93 mm s^?1 were created using a 1.6-mm diameter syringe. We tested the effects of sparger velocity over a range of 2.48 to 73.4 m s^?1 and the effects of bubble size by using two styles of air stones and an open glass pipette, which created a bubble sizes in the range of 0.5 to 5 mm. We calculated oxygen mass transfer coefficient, k_La, values for all experimental conditions. Cell growth increased linearly with increased superficial gas velocity and decreased with increased sparger velocity. Results indicated that smaller bubble size leads to some initial cell damage, but after time, the increased gas transfer as reflected by the k_La value produced higher growth than larger bubbles. Two mechanisms were observed to correlate with cell damage in T. lutea: increasing velocity at the sparger tip and bubble bursting at the surface. These results demonstrate a method to test sensitivity of T. lutea to aeration, which is important for the design of airlift systems.     

Local gas hydrodynamics in an external-loop airlift reactor: newtonian and non-newtonian fluids

Measurements of local gas phase characteristics are obtained in an external-loop airlift reactor filled with newtonian or viscous non-newtonian liquids. A double-optical fiber probe technique is used. It allows the determination of the axial and radial profiles of gas hold-up, bubbling frequency, bubble size and velocity. In the case of air-water system, the results show a strong effect of radial liquid velocity variation on the gas flow characteristics at the bottom of the riser. In the case of highly viscous non-newtonian solution, the gas flow is strongly affected by the gas distribution just above the gas sparger. This study also points out the bubble coalescence and the break-up phenomena in different liquids and levels in the reactor. Furthermore, the local measurements of bubble size and velocity allows to gain more detailed information on the dynamics of the bubble-flow and shows a tendency of large bubbles to circulate in the column center.     

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.     

The secretion of inert gas into the swim-bladder of fish

The composition of the gas mixture secreted into the swim-bladders of several species of fish has been determined in the mass spectrometer. The secreted gas differed greatly from the gas mixture breathed by the fish in the relative proportions of the chemically inert gases, argon, neon, helium, and nitrogen. Relative to nitrogen the proportion of the very soluble argon was increased and the proportions of the much less soluble neon and helium decreased. The composition of the secreted gas approaches the composition of the gas mixture dissolved in the tissue fluid. A theory of inert gas secretion is proposed. It is suggested that oxygen gas is actively secreted and evolved in the form of minute bubbles, that inert gases diffuse into these bubbles, and that the bubbles are passed into the swim-bladder carrying with them inert gases. Coupled to a preferential reabsorption of oxygen from the swim-bladder this mechanism can achieve high tensions of inert gas in the swim-bladder. The accumulation of nearly pure nitrogen in the swim-bladder of goldfish (Carassius auratus) is accomplished by the secretion of an oxygen-rich gas mixture followed by the reabsorption of oxygen.     

Effect of gas pressure in the root and stem base zone on methane transport through rice bodies

Ebullition of gas bubbles from the soil surface is, in some cases (e.g., in early growth stage of rice), one of the major pathways for methane transport from rice paddies to the atmosphere. However, the role of the gas phase (entrapped gas) in the paddy soil in plant-mediated methane transport, which is the major pathway for methane emission, has not been clarified. To clarify the effect of the gas phase below ground on the methane emission rate through rice plants, we partly exposed the root and stem base of hydroponically grown rice to a high concentration of methane gas at various gas pressures, and immersed the rest of the roots in a solution with a high methane concentration. The methane emission rate was measured from the top of the rice plant using a flow-through chamber method. The methane emission rate drastically increased with a small increase in gas pressure in the gas phase at the root and stem base zone, with about a 3 times larger emission rate being observed with 10 × 10^-3 atm of extra pressure (corresponding to 10 cm of standing water in rice paddy) compared to no extra pressure. However, when alginate was applied to the stem near the base to prevent contact with the gas phase, the methane emission rate did not increase with increasing gas pressure. On the other hand, from observations in the rice paddy, it was found that the gas is entrapped near the surface (e.g., at a depth of 1 cm) and the gas entrapped in the soil would come into direct contact with a part of the stem near the base of the rice plant. Thus, the gas entrapped in the soil could enter into the rice body directly from the part of the stem near the base which is beneath the soil surface due to gas pressure in the gas phase resulting from the pressure exerted by the standing water. Hence, this mechanism involving the entrapped gas could play an important role in methane emission from rice paddy by affecting the plant-mediated methane transport as well as ebullition of gas bubbles.     

Microflotation: New low gas-flow rate foam separation technique for bacteria and algae

Foam separation of microorganisms has been investigated with varying success by many workers, usually at high rates of gas flow. Microflotation was developed to overcome some of the disadvantages inherent in these high gas-flow-rate processes and is introduced in this paper as a new technique for the foam separation of microorganisms at low gas-flow rates. With microflotation, a stable surface phase is produced by adding an insoluble collector such as a long-chain fatty acid or amine. The formation of an insoluble surface phase eliminates the need for high foaming. Low rates of gas flow are used resulting in a more efficient separation and a less voluminous and drier surface phase upon which to collect the microorganisms. The efficiency of this technique is also improved by using flotation aids such as frothers and flocculents. Frothers are used to improve the collector properties of the surfactant and to refine further the small bubbles produced by a very fine sparger. Small concentrations of flocculents, such as alum, are used to partially agglomerate the organisms and provide sites for adsorption of collector. The work described in this paper is preliminary in nature, designed to illustrate that a low flow-rate process may be used to separate microorganisms and to stimulate further research. The applications discussed are removal of the bacterium, Escherichia coli, and alum, and two species of algae, Chlamydomonas reinhardtii and Chlorella ellipsoidea, using stearylamine without alum. The frother used was ethanol.     

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 %.     

Effects of bubble–liquid two‐phase turbulent hydrodynamics on cell damage in sparged bioreactor

According to recent experimental studies on sparged bioreactors, significant cell damage may occur at the gas inlet region near the sparger. Although shear stress was proposed to be one of the potential causes for cell damage, detailed hydrodynamic studies at the gas inlet region of gas–liquid bioreactors have not been performed to date. In this work, a second‐order moment (SOM) bubble–liquid two‐phase turbulent model based on the two‐fluid continuum approach is used to investigate the gas–liquid hydrodynamics in the bubble column reactor and their potential impacts on cell viability, especially at the gas inlet region. By establishing fluctuation velocity and bubble–liquid two‐phase fluctuation velocities correlation transport equations, the anisotropy of two‐phase stresses and the bubble–liquid interactions are fully considered. Simulation results from the SOM model indicate that shear and normal stresses, turbulent energy dissipation rate, and the turbulent kinetic energy are generally smaller at the gas inlet region when compared with those in the fully developed region. In comparison, a newly proposed correlation expression, stress‐induced turbulent energy production (STEP), is found to correlate well with the unusually high cell death rate at the gas inlet region. Therefore, STEP, which represents turbulent energy transfer to a controlled volume induced by a combination of shear and normal stresses, has the potential to provide better explanation for increased cell death at the sparger region. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:48–58, 2014     

CFD and kinetic-based modeling to optimize the sparger design of a large-scale photobioreactor for scaling up of biofuel production

Microalgal biofuels have not yet achieved wide-spread commercialization, partially as a result of the complexities involved with designing and scaling up of their biosystems. The sparger design of a pilot-scale photobioreactor (120 L) was optimized to enable the scale-up of biofuel production. An integrated model coupling computational fluid dynamics and microalgal biofuel synthesis kinetics was used to simulate the biomass growth and novel biofuel production (i.e., bisabolene) in the photobioreactor. Bisabolene production from Chlamydomonas reinhardtii mutant was used as an example to test the proposed model. To select the optimal sparger configuration, a rigorous procedure was followed by examining the effects of sparger design parameters (number and diameter of sparger holes and gas flow rates) on spatially averaged bubble volume fraction, light intensity, friction velocity, power input, biomass concentration, and bisabolene production. The optimized sparger design increases the final biomass concentration by 18%, thereby facilitating the scaling up of biofuel production.     

NS0 cell damage by high gas velocity sparging in protein-free and cholesterol-free cultures

Recent developments in high cell density and high productivity fed-batch animal cell cultures have placed a high demand on oxygenation and carbon dioxide removal in bioreactors. The high oxygen demand is often met by increasing agitation and sparging rates of air/O2 in the bioreactors. However, as we demonstrate in this study, an increase of gas sparging can result in cell damage at the sparger site due to high gas entrance velocities. Previous studies have showed that gas bubble breakup at the culture surface was primarily responsible for cell damage in sparged bioreactors. Such cell damage can be reduced by use of surfactants such as Pluronic F-68 in the culture. In our results, where NS0 cells were grown in a protein-free and cholesterol-free medium containing 0.5 g/L Pluronic F-68, high gas entrance velocity at the sparger site was observed as the second mechanism for cell damage. Experiments were performed in scaled-down spinners to model the effect of hydrodynamic force resulting from high gas velocities on antibody-producing NS0 cells. Cell growth and cell death were described by first-order kinetics. Cell death rate constant increased significantly from 0.04 to 0.18 day(-1) with increasing gas entrance velocity from 2.3 to 82.9 m/s at the sparger site. The critical gas entrance velocity for the NS0 cell line studied was found to be approximately 30 m/s; velocities greater than 30 m/s caused cell damage which resulted in reduced viability and consequently reduced antibody production. Observations from a second cholesterol-independent NS0 cell line confirmed the occurrence of cell damage due to high gas velocities. Increasing the concentration of Pluronic F-68 from 0.5 to 2 g/L had no additional protective effect on cell damage associated with high gas velocity at the sparger. The results of gas velocity analysis for cell damage have been applied in two case studies of large-scale antibody manufacturing. The first is a troubleshooting study for antibody production carried out in a 600 L bioreactor, and the second is the development of a gas sparger design for a large bioreactor scale (e.g., 10,000 L) for antibody manufacturing.     

Observation and quantification of gas bubble formation on a mechanical heart valve

Clinical studies using transcranial Doppler ultrasonography in patients with mechanical heart valves (MHV) have detected gaseous emboli. The relationship of gaseous emboli release and cavitation on MHV has been a subject of debate in the literature. To study the influence of cavitation and gas content on the formation and growth of stable gas bubbles, a mock circulatory loop, which employed a Medtronic-Hall pyrolytic carbon disk valve in the mitral position, was used. A high-speed video camera allowed observation of cavitation and gas bubble release on the inflow valve surfaces as a function of cavitation intensity and carbon dioxide (CO2) concentration, while an ultrasonic monitoring system scanned the aortic outflow tract to quantify gas bubble production by calculating the gray scale levels of the images. In the absence of cavitation, no stable gas bubbles were formed. When gas bubbles were formed, they were first seen a few milliseconds after and in the vicinity of cavitation collapse. The volume of the gas bubbles detected in the aortic track increased with both increased CO2 and increased cavitation intensity. No correlation was observed between O2 concentration and bubble volume. We conclude that cavitation is an essential precursor to stable gas bubble formation, and CO2, the most soluble blood gas, is the major component of stable gas bubbles.