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

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

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

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

Macromixing characteristics of gas-liquid jet loop reactors

Measurements of liquid macromixing characteristics are reported for a half industrial scaled jet loop reactor operating with air-water mixtures. Based on a model of loop reactors with sections of different mixing behavior the single circulation dispersion coefficient can be split into its components caused by the riser and the downcomer. The dispersion coefficient of the riser is about 100 times greater than that of the downcomer. The addition of gas involves greater dispersions coefficients. The comparison of the mixing times of the JLR with those of stirred vessels leads to the conclusion that the JLR is equivalent or even superior to stirred vessels.     

Computational fluid dynamics modeling of an inverted frustoconical shaking bioreactor for mammalian cell suspension culture

We previously developed an inverted frustoconical shaking bioreactor (IFSB) which had high mammalian cell culture performance when compared with a mechanically stirred tank reactor (STR) or a flat-bottom shaking bioreactor (FBSB). Here, we determined the mixing time (t) and volumetric oxygen transfer coefficient (k _La) of this IFSB at various speeds, and simulated the fluid hydrodynamics, including the shear stress and specific surface area, by computational fluid dynamics. The shortest mixing time was observed in a STR. The maximum kLa value of 12/h was achieved in the IFSB at an aeration rate of 4 L/h, demonstrating that our IFSB has enhanced oxygen transfer capabilities needed to meet the demands of mammalian cells. Simulation studies revealed a 3% greater specific surface area and a 21% lower shear strain in the IFSB compared to an FBSB under the same conditions. Additionally, the conical angle of the vessel, which significantly affected cell growth and recombinant protein production, was tested here. We conclude that, compared to the STR and FBSB, the IFSB has an increased liquid surface area for oxygen uptake and exhaust CO₂ stripping, an enhanced k _La for cell robust growth to a high cell density, and a lower shear stress to alleviate cell damage.     

Jet aeration as alternative to overcome mass transfer limitation of stirred bioreactors

In industrial biotechnology increasing reactor volumes have the potential to reduce production costs. Whenever the achievable space time yield is determined by the mass transfer performance of the reactor, energy efficiency plays an important role to meet the requirements regarding low investment and operating costs. Based on theoretical calculations, compared to bubble column, airlift reactor, and aerated stirred tank, the jet loop reactor shows the potential for an enhanced energetic efficiency at high mass transfer rates. Interestingly, its technical application in standard biotechnological production processes has not yet been realized. Compared to a stirred tank reactor powered by Rushton turbines, maximum oxygen transfer rates about 200% higher were achieved in a jet loop reactor at identical power input in a fed batch fermentation process. Moreover, a model‐based analysis of yield coefficients and growth kinetics showed that E. coli can be cultivated in jet loop reactors without significant differences in biomass growth. Based on an aerobic fermentation process, the assessment of energetic oxygen transfer efficiency [kgO₂ kW^?1 h^?1] for a jet loop reactor yielded an improvement of almost 100%. The jet loop reactor could be operated at mass transfer rates 67% higher compared to a stirred tank. Thus, an increase of 40% in maximum space time yield [kg m^?3 h^?1] could be observed.     

Gas–liquid mass transfer in an up-flow cocurrent packed-bed biofilm reactor

Gas–liquid mass transfer was investigated in an up-flow cocurrent packed-bed biofilm reactor. In aerobic processes gas–liquid mass transfer can be considered as a key operational parameter as well as in reactor scale-up. The present paper investigates the influence of the liquid phase mixing in the determination of the volumetric gas–liquid mass transfer coefficient (k_La) coefficient. Residence time distribution (RTD) experiments were performed in the reactor to determine the flow pattern of the liquid phase and to model mathematically the liquid phase mixing. The mathematical model derived from RTD experiments was used to evaluate the influence of the liquid mixing on the experimental estimation of the (k_La) in this reactor type. The methods used to estimate the k_La coefficient were: (i) dynamic gassing-out, (ii) sulphite method, and (iii) in-process estimation through biological conversion obtained in the reactor. The use of standard chemical engineering correlations to determine the k_La in this type of bioreactors is assessed. Experimental and modelling results show how relevant can be to take into consideration the liquid phase mixing in the calculations of the most-used methods for the estimation of k_La coefficient. k_La coefficient was found to be strongly heterogeneous along the reactor vertical axis. The value of the k_La coefficient for the packed-bed section ranged 0.01–0.12 s⁻¹. A preliminary correlation was established for up-flow cocurrent packed-bed biofilm reactors as a function of gas superficial velocity.     

The scale-up of plant cell culture: Engineering considerations

The enormous versatility of plants has continued to provide the impetus for the development of plant tissue culture as a commercial production strategy for secondary metabolites. Unfortunately problems with slow growth rates and low products yields, which are generally non-growth associated and intracellular, have made plant cell culture-based processes, with a few exceptions, economically unrealistic. Recent developments in reactor design and control, elicitor technology, molecular biology, and consumer demand for natural products, are fuelling a renaissance in plant cell culture as a production strategy. In this review we address the engineering consequences of the unique characteristics of plant cells on the scale-up of plant cell culture.Abbreviations a gas-liquid interfacial area per volume - C dissolved oxygen concentration - C^* liquid phase oxygen concentration in equilibrium with the partial pressure of oxygen in the bulk gas phase - K_L overall mass transfer coefficient - k_L liquid film mass transfer coefficient - m_O2 cell maintenance coefficient for oxygen - OTR oxygen transfer rate - OUR oxygen uptake rate - pO₂ partial pressure of oxygen - STR stirred-tank reactor - v.v.m. volume of gas fed per unit operating volume of reactor per minute - X biomass concentration - Y_x/O2 biomass yield coefficient for oxygen - specific growth rate     

Utilizing the tubular bioreactor for continuous recovery of secreted fusion protein from recombinant Escherichia coli

In order to improve the cultivation properties of a traditional continuous stirred tank reactor (CSTR), we introduced a circulation unit made of four inorganic membranes in stainless steel tubes in parallel configuration, the so-called Tubular Bioreactor (TBR). Furthermore, the TBR outlet tube, which has a restriction nozzle at the end, was installed on top of the fermentor vessel, thereby creating a strong jet flow into the reactor and thus improving the mixing and the oxygen transfer rate. The k _La could be increased by approximately 50%. This setup was used for cultivations of recombinant Escherichia coli in a minimal medium and high cell density. More than 50 g dry cell mass/dm³ was obtained. Simultaneously, we have produced an elongated form of human insulin-like growth factor II, which was a secreted fusion protein utilizing the E. coli secretion system based on staphylococcus protein A. The product could be recovered continuously through the TBR-membrane.     

Mass transfer and liquid mixing in an airlift reactor with a net draft tube

Mass transfer and liquid mixing in an airlift reactor with a net draft tube were experimentally investigated. Four different column diameters were considered. The mass transfer was measured using the volumetric gas-liquid mass transfer coefficient which was determined by the dynamic method. The mass transfer coefficients in the airlift reactors with different column diameters were not always higher than those in the bubble columns. The liquid mixing was measured using mixing time which was determined by a pulse technique. Under the same superficial gas velocity, the mixing times of the airlift reactors with a net draft tube were always less than those of the bubble columns.List of Symbols C mol·dm^–3 bulk concentration of dissolved oxygen - C ₀ mol·dm^–3 initial concentration of dissolved oxygen - C _e mol·dm^–3 saturated concentration of dissolved oxygen - ¯C dimensionless dissolved oxygen concentration - D _c cm diameter of column - D _N cm diameter of the nozzle hole - D _T cm diameter of the net draft tube - H _L cm static liquid height - H _T cm height of the net draft tube - k _L a hr^–1 volumetric mass transfer coefficient - L _T cm length of the net draft tube - t _M sec mixing time of the liquid phase - t ₀ sec mixing time of the liquid phase in a bubble column - V _L dm³ volume of the liquid phase - U _g cm/s superficial air velocity     

A scale-down two-compartment reactor with controlled substrate oscillations: Metabolic response of Saccharomyces cerevisiae

Substrate concentration gradients are likely to appear during large scale fermentations. To study effects of such gradients on microorganisms, an aerated scale-down reactor system was constructed. It consists of a plug flow reactor (PFR) and a stirred tank reactor (STR), between which the medium is circulated. The PFR, which is an aerated static mixer reactor, was characterized with respect to plug flow behaviour and oxygen transfer. A Bodenstein number of 15–220, depending on residence time and aeration rate, and a k_La of 500–1130 h^–1, depending mainly on aeration rate, were obtained. The biological test system used, was aerobic ethanol production by Saccharomyces cerevisiae, due to sugar excess. The ethanol concentration profile and the yield of biomass were compared in two fed-batch fermentations. In the first case, the feeding point of molasses was located at the inlet of the PFR. This simulates location of the feeding point in the segregated part of a heterogeneous reactor, with local high sugar concentrations. In the second mode of operation, as a control with good mixing conditions, the PFR was disconnected from the STR, into which the substrate was fed. Differences were found: Up to 6% less biomass was produced and a larger amount of ethanol was formed in the two-compartment reactor system, due to the uneven sugar concentration distribution. This emphasizes the importance of the location of, and the mixing conditions at, the feeding point in a bioreactor.     

Application of a two-dimensional disposable rocking bioreactor to bacterial cultivation for recombinant protein production

Disposable rocking bioreactors (RBs) are widely employed for cultivation of recombinant mammalian and insect cell lines, although the perception of inadequate mass transfer has prevented their application to bioprocesses based on microbial platforms. In this study, one-dimensional (1D) and two-dimensional (2D) RBs were assessed and compared with the conventional stirred tank reactor (STR) for recombinant therapeutic protein production in Escherichia coli. The comparison involved: (1) physical characterization of oxygen mass transfer efficiency and mixing intensity, (2) growth characteristics in batch cultivation, and (3) culture performance for the production of recombinant protein. Our results show that oxygen mass transfer was comparable between the 1D RB and STR at low working volume (WV), declining linearly with increasing WV, and was highest in the 2D RB for all tested WVs with the maximum mass transfer coefficient (k_La) at 3 L WV. Well mixing behavior was observed in all three systems for water and aqueous carboxymethylcellulose (CMC) solutions. Batch growth characteristics were similar in all bioreactor systems, although metabolite accumulation was significant in the 1D RB. Culture performance for the production of recombinant GST-hCD83ext (glutathione S-transferase-hCD83ext fusion protein) was similar in terms of soluble protein yield and inclusion body formation for all bioreactor systems.     

Comparison of single- and three-stage tower loop reactors

Summary Hansenula polymorpha was cultured for long periods in 254 cm high single and three-stage countercurrent tower loop reactors 20 cm in diameter using ethanol as a substrate in the absence and presence of antifoam agents (Desmophen 3600 and/or soy oil). In the absence of antifoam agents in the three-stage column, much higher volumetric mass transfer coefficients were attained than in the corresponding single-stage column. The cell productivity in the former, however, was only slightly higher than in the single-stage column due to considerable enrichment of the cells in the foam and nonuniform cell concentration distribution in the three-stage column. In the presence of antifoam agents the three-stage column has a higher cell productivity, OTR, k_L a and a lower specific energy requirement with regard to the absorbed oxygen and/or produced cell mass than the single stage column. The reactor performance is especially high if the bubbling layer height is reduced to 20 cm. Soy oil has considerably less foam eliminating property than Desmophen. Since the soy oil is metabolized by the yeast, large amounts are needed to operate these reactors.     

Venturi aeration and thermophilic aerobic sewage sludge digestion in small-scale reactors

Summary The aeration performance of two venturi aeration reactors (operating volumes 381 and 251) was studied for an air-water system. It was found that the mass transfer coefficient (k _la) could be described in terms of the superficial gas velocity (V _s) alone by the simple expressionk _La=aV _infb ^supS with constantsa=0.313,b=0.579 for the 38-1 reactor anda=0.214,b=0.534 for the 25-1 reactor. A similar relationship was obeyed when the 38-1 reactor was aerated with a diffuser tile (a=17.0,b=1.52). A linear relationship betweenk _La and gas hold-up was observed for the 38-1 reactor with both venturi and diffuser aeration. The 25-1 reactor was used successfully for the thermophilic aerobic digestion of sewage sludge. A mean sludge temperature rise of 30°C was observed. Chemical oxygen demand, pH, and total solids content of the digested sludge differed significantly from the feed sludge and were similar to values obtained for full-scale thermophilic aerobic digestion. No significant differences between inorganic solids content, dissolved oxygen concentration, or redox potential were observed between feed and digested sludge.     

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 transfer characteristics of a centrifugal, packed-bed reactor during viscous xanthan fermentation

The oxygen transfer properties of a novel, centrifugal, packed-bed reactor (CPBR) during viscous xanthan fermentation were determined with respect to the effects of the arrangement of the centrifugal, packed bed (CPB) and the recirculation loop (RL). Characterized by the maximum volumetric transfer coefficient (k_La) in xanthan broth, the aeration efficiency of CPBR was compared to those in stirred-tank reactors (STR) equipped with disc turbines (DT) or marine propellers (MP), and to that in a water-in-oil emulsion (WIO). As expected, STR-WIO showed the highest k_La (0.038 s^-1 at 2%) among all systems studied due to reduced broth viscosity; however, practical difficulties exist in product recovery. It was found that, at 3.5% xanthan the k_La in CPBR (0.018 s^-1) was higher than that of STR (0.005 s^-1) and close to that of STR-WIO (0.020 s^-1), indicating improved oxygen transfer at such a xanthan concentration. The exterior baffles along the rotating fibrous matrix offer additional agitation in the viscous broth. A gas-continuous arrangement, in which the CPB was kept above the broth, was able to elevate k_La to 0.023 s^-1, higher than that of STR-WIO. The external RL operated by a peristaltic pump was found to play an important role in CPBR aeration by providing better gas-liquid contact. With the improved oxygen transfer efficiency in CPBR at high xanthan concentrations, the CPBR system is practically the preferred system for xanthan fermentation. The characteristic roles of CPB arrangement and the RL should be considered primarily during scale-up operation.     

Influence of dissolved oxygen on the nitrification kinetics in a circulating bed biofilm reactor

The influence of dissolved oxygen concentration on the nitrification kinetics was studied in the circulating bed reactor (CBR). The study was partly performed at laboratory scale with synthetic water, and partly at pilot scale with secondary effluent as feed water. The nitrification kinetics of the laboratory CBR as a function of the oxygen concentration can be described according to the half order and zero order rate equations of the diffusion-reaction model applied to porous catalysts. When oxygen was the rate limiting substrate, the nitrification rate was close to a half order function of the oxygen concentration. The average oxygen diffusion coefficient estimated by fitting the diffusion-reaction model to the experimental results was around 66% of the respective value in water. The experimental results showed that either the ammonia or the oxygen concentration could be limiting for the nitrification kinetics. The latter occurred for an oxygen to ammonia concentration ratio below 1.5–2 gO₂/gN-NH₄ ⁺ for both laboratory and pilot scale reactors. The volumetric oxygen mass transfer coefficient (k _L a) determined in the laboratory scale reactor was 0.017?s^?1 for a superficial air velocity of 0.02?m s^?1, and the one determined in the pilot scale reactor was 0.040?s^?1 for a superficial air velocity of 0.031?m?s^?1. The k _L a for the pilot scale reactor did not change significantly after biofilm development, compared to the value measured without biofilm.     

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.     

Polysialic acid production using Escherichia coli K1 in a disposable bag reactor

Polysialic acid (polySia), consisting of α‐(2,8)‐linked N‐acetylneuraminic acid monomers plays a crucial role in many biological processes. This study presents a novel process for the production of endogenous polySia using Escherichia coli K1 in a disposable bag reactor with wave‐induced mixing. Disposable bag reactors provide easy and fast production in terms of regulatory requirements as GMP, flexibility, and can easily be adjusted to larger production capacities not only by scale up but also by parallelization. Due to the poor oxygen transfer rate compared to a stirred tank reactor, pure oxygen was added during the cultivation to avoid oxygen limitation. During the exponential growth phase the growth rate was 0.61 h^?1. Investigation of stress‐related product release from the cell surface showed no significant differences between the disposable bag reactor with wave‐induced mixing and the stirred tank reactor. After batch cultivation a cell dry weight of 6.8 g L^?1 and a polySia concentration of 245 mg L^?1 were reached. The total protein concentration in the supernatant was 132 mg L^?1. After efficient and time‐saving downstream processing characterization of the final product showed a protein content of below 0.04 mg_protein/g_polySia and a maximal chain length of ～90 degree of polymerization.     

Novel electrodynamic oscillation technique enables enhanced mass transfer and mixing for cultivation in micro-bioreactor

Micro-bioreactors (MBRs) have become an indispensable part for modern bioprocess development enabling automated experiments in parallel while reducing material cost. Novel developments aim to further intensify the advantages as dimensions are being reduced. However, one factor hindering the scale-down of cultivation systems is to provide adequate mixing and mass transfer. Here, vertical oscillation is demonstrated as an effective method for mixing of MBRs with a reaction volume of 20 μL providing adequate mass transfer. Electrodynamic exciters are used to transduce kinetic energy onto the cultivation broth avoiding additional moving parts inside the applied model MBR. The induced vertical vibration leads to oscillation of the liquid surface corresponding to the frequency and displacement. On this basis, the resonance frequency of the fluid was identified as the most decisive factor for mixing performance. Applying this vertical oscillation method outstanding mixing times below 1 s and exceptionally high oxygen transport with volumetric mass transfer coefficients (k_La) above 1,000/hr can be successfully achieved and controlled. To evaluate the applicability of this vertical oscillation mixing for low volume MBR systems, cultivations of Escherichia coli BL21 as proof-of-concept were performed. The dissolved oxygen was successfully online monitored to assure any avoidance of oxygen limitations during the cultivation. The here presented data illustrate the high potential of the vertical oscillation technique as a flexible measure to adapt mixing times and oxygen transfer according to experimental demands. Thus, the mixing technique is a promising tool for various biological and chemical micro-scale applications still enabling adequate mass transfer.     

Correlation between mass transfer coefficient k<Subscript>L</Subscript>a and relevant operating parameters in cylindrical disposable shaken bioreactors on a bench-to-pilot scale

Background

Among disposable bioreactor systems, cylindrical orbitally shaken bioreactors show important advantages. They provide a well-defined hydrodynamic flow combined with excellent mixing and oxygen transfer for mammalian and plant cell cultivations. Since there is no known universal correlation between the volumetric mass transfer coefficient for oxygen k_La and relevant operating parameters in such bioreactor systems, the aim of this current study is to experimentally determine a universal k_La correlation.

Results

A Respiration Activity Monitoring System (RAMOS) was used to measure k_La values in cylindrical disposable shaken bioreactors and Buckingham’s π-Theorem was applied to define a dimensionless equation for k_La. In this way, a scale- and volume-independent k_La correlation was developed and validated in bioreactors with volumes from 2 L to 200 L. The final correlation was used to calculate cultivation parameters at different scales to allow a sufficient oxygen supply of tobacco BY-2 cell suspension cultures.

Conclusion

The resulting equation can be universally applied to calculate the mass transfer coefficient for any of seven relevant cultivation parameters such as the reactor diameter, the shaking frequency, the filling volume, the viscosity, the oxygen diffusion coefficient, the gravitational acceleration or the shaking diameter within an accuracy range of +/? 30%. To our knowledge, this is the first k_La correlation that has been defined and validated for the cited bioreactor system on a bench-to-pilot scale.