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     

Prediction of gas-liquid mass transfer coefficient in sparged stirred tank bioreactors

Oxygen mass transfer in sparged stirred tank bioreactors has been studied. The rate of oxygen mass transfer into a culture in a bioreactor is affected by operational conditions and geometrical parameters as well as the physicochemical properties of the medium (nutrients, substances excreted by the micro-organism, and surface active agents that are often added to the medium) and the presence of the micro-organism. Thus, oxygen mass transfer coefficient values in fermentation broths often differ substantially from values estimated for simple aqueous solutions. The influence of liquid phase physicochemical properties on kLa must be divided into the influence on k(L) and a, because they are affected in different ways. The presence of micro-organisms (cells, bacteria, or yeasts) can affect the mass transfer rate, and thus kLa values, due to the consumption of oxygen for both cell growth and metabolite production. In this work, theoretical equations for kLa prediction, developed for sparged and stirred tanks, taking into account the possible oxygen mass transfer enhancement due to the consumption by biochemical reactions, are proposed. The estimation of kLa is carried out taking into account a strong increase of viscosity broth, changes in surface tension and different oxygen uptake rates (OURs), and the biological enhancement factor, E, is also estimated. These different operational conditions and changes in several variables are performed using different systems and cultures (xanthan aqueous solutions, xanthan production cultures by Xanthomonas campestris, sophorolipids production by Candida bombicola, etc.). Experimental and theoretical results are presented and compared, with very good results.     

A restructured framework for modeling oxygen transfer in two-phase partitioning bioreactors

This communication proposes a mechanistic modification to a recently published method for analyzing oxygen mass transfer in two-phase partitioning bioreactors (Nielsen et al., 2003), and corrects an oversight in that paper. The newly proposed modification replaces the earlier empirical approach, which treated the two liquid phases as a single, homogeneous liquid phase, with a two-phase mass transfer model of greater fundamental rigor. Additionally, newly developed empirical models are presented that predict the mass transfer coefficient of oxygen absorption in both aqueous medium and an organic phase (n-hexadecane) as a function of bioreactor operating conditions. Experimental values and theoretical predictions of mass transfer coefficients in two-phase dispersions, k(L)a(TP), are compared. The revised approach more clearly demonstrates the potential for oxygen mass transfer enhancement by organic phase addition, one of the motivations for employing a distinct second phase in a partitioning bioreactor.     

Development of a method for reliable power input measurements in conventional and single‐use stirred bioreactors at laboratory scale

Power input is an important engineering and scale‐up/down criterion in stirred bioreactors. However, reliably measuring power input in laboratory‐scale systems is still challenging. Even though torque measurements have proven to be suitable in pilot scale systems, sensor accuracy, resolution, and errors from relatively high levels of friction inside bearings can become limiting factors at smaller scales. An experimental setup for power input measurements was developed in this study by focusing on stainless steel and single‐use bioreactors in the single‐digit volume range. The friction losses inside the air bearings were effectively reduced to less than 0.5% of the measurement range of the torque meter. A comparison of dimensionless power numbers determined for a reference Rushton turbine stirrer (N_P = 4.17 ± 0.14 for fully turbulent conditions) revealed good agreement with literature data. Hence, the power numbers of several reusable and single‐use bioreactors could be determined over a wide range of Reynolds numbers between 100 and >10⁴. Power numbers of between 0.3 and 4.5 (for Re = 10⁴) were determined for the different systems. The rigid plastic vessels showed similar power characteristics to their reusable counterparts. Thus, it was demonstrated that the torque‐based technique can be used to reliably measure power input in stirred reusable and single‐use bioreactors at the laboratory scale.     

A novel method of simulating oxygen mass transfer in two-phase partitioning bioreactors

An empirical correlation, based on conventional forms, has been developed to represent the oxygen mass transfer coefficient as a function of operating conditions and organic fraction in two-phase, aqueous-organic dispersions. Such dispersions are characteristic of two-phase partitioning bioreactors, which have found increasing application for the biodegradation of toxic substrates. In this work, a critical distinction is made between the oxygen mass transfer coefficient, k(L)a, and the oxygen mass transfer rate. With an increasing organic fraction, the mass transfer coefficient decreases, whereas the oxygen transfer rate is predicted to increase to an optimal value. Use of the correlation assumes that the two-phase dispersion behaves as a single homogeneous phase with physical properties equivalent to the weighted volume-averaged values of the phases. The addition of a second, immiscible liquid phase with a high solubility of oxygen to an aqueous medium increases the oxygen solubility of the system. It is the increase in oxygen solubility that provides the potential for oxygen mass transfer rate enhancement. For the case studied in which n-hexadecane is selected as the second liquid phase, additions of up to 33% organic volume lead to significant increases in oxygen mass transfer rate, with an optimal increase of 58.5% predicted using a 27% organic phase volume. For this system, the predicted oxygen mass transfer enhancements due to organic-phase addition are found to be insensitive to the other operating variables, suggesting that organic-phase addition is always a viable option for oxygen mass transfer rate enhancement.     

Experimental and computational characterization of mass transfer in high turndown bioreactors

Single-use bioreactors (SUBs, or disposable bioreactors) are extensively used for the clinical and commercial production of biologics. Despite widespread application, minimal results have been reported utilizing the turndown ratio; an operation mode where the working range of the bioreactor can be expanded to include low fluid volumes. In this work, a systematic investigation into free surface mass transfer and cell growth in high turndown single-use bioreactors is presented. This approach, which combines experimental mass transfer measurements with numerical simulation, deconvolutes the combined effects of headspace mixing and the free surface convective mass transfer on cell growth. Under optimized conditions, mass transfer across the interface alone may be sufficient to satisfy oxygen demands of the cell culture. Within the context of high turndown bioreactors, this finding provides a counterpoint to traditional sparge-based bioreactor operational philosophy. Multiple monoclonal antibody-producing cell lines grown using this high turndown approach showed similar viable cell densities to those cells expanded using a traditional cell bag rocker. Furthermore, cells taken directly from the turndown expansion and placed into production showed identical growth characteristics to traditionally expanded cultures. Taken together, these results suggest that the Xcellerex SUB can be run at a 5:1 working volume as a seed to itself, with no need for system modifications, potentially simplifying preculture operations.     

Promotion of oxygen transfer in three-phase fluidized-bed bioreactors by floating bubble breakers

The objective of the present study was to investigate a method to enhance the volumetric rate of oxygen transfer in three-phase fluidized-bed bioreactors. The rates of oxygen transfer from air bubbles to viscous liquid media were promoted by floating bubble breakers in three-phase fluidized beds operated in the bubble coalescing regime. The liquid-phase volumetric oxygen transfer coefficient has been recovered by fitting the axial dispersion model to the resultant data, and its dependence on the experimental variables, such as the gas and liquid flow rates, particle size, concentration of bubble breakers, and liquid viscosity, has been examined. The results indicate that the liquid-phase volumetric oxygen transfer coefficient can be enhanced up to 20-25%. The coefficient exhibits a maximum with respect to the volume ratio of the floating bubble breakers to the fluidized solid particles; it increases with increases in the gas and liquid flow rates and size of fluidized particles, while it decreases with an increase in the liquid viscosity. An expression has been developed to correlate the liquid-phase volumetric oxygen transfer coefficient with the experimental variables.     

Assessment of mass transfer and mixing in rigid lab‐scale disposable bioreactors at low power input levels

Mass transfer, mixing times and power consumption were measured in rigid disposable stirred tank bioreactors and compared to those of a traditional glass bioreactor. The volumetric mass transfer coefficient and mixing times are usually determined at high agitation speeds in combination with sparged aeration as used for single cell suspension and most bacterial cultures. In contrast, here low agitation speeds combined with headspace aeration were applied. These settings are generally used for cultivation of mammalian cells growing adherent to microcarriers. The rigid disposable vessels showed similar engineering characteristics compared to a traditional glass bioreactor. On the basis of the presented results appropriate settings for adherent cell culture, normally operated at a maximum power input level of 5 W m^?3, can be selected. Depending on the disposable bioreactor used, a stirrer speed ranging from 38 to 147 rpm will result in such a power input of 5 W m^?3. This power input will mix the fluid to a degree of 95% in 22 ± 1 s and produce a volumetric mass transfer coefficient of 0.46 ± 0.07 h^?1. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1269–1276, 2014     

The baffled microtiter plate: Increased oxygen transfer and improved online monitoring in small scale fermentations

Most experiments in screening and process development are performed in shaken bioreactors. Today, microtiter plates are the preferred vessels for small‐scale microbial cultivations in high throughput, even though they have never been optimized for this purpose. To interpret the experimental results correctly and to obtain a base for a meaningful scale‐up, sufficient oxygen supply to the culture liquid is crucial. For shaken bioreactors this problem can generally be addressed by the introduction of baffles. Therefore, the focus of this study is to investigate how baffling and the well geometry affect the maximum oxygen transfer capacity (OTR_max) in microtiter plates. On a 48‐well plate scale, 30 different cross‐section geometries of a well were studied. It could be shown that the introduction of baffles into the common circular cylinder of a microtiter plate well doubles the maximum oxygen transfer capacity, resulting in values above 100 mmol/L/h (k_La > 600 1/h). To also guarantee a high volume for microbial cultivation, it is important to maximize the filling volume, applicable during orbital shaking. Additionally, the liquid height at the well bottom was examined, which is a decisive parameter for online‐monitoring systems such as the BioLector. This technology performs fiber‐optical measurements through the well bottom, therefore requires a constant liquid height at all shaking frequencies. Ultimately, a six‐petal flower‐shaped well geometry was shown to be the optimal solution taking into account all aforementioned criteria. With its favorable culture conditions and the possibility for unrestricted online monitoring, this novel microtiter plate is an efficient tool to gain meaningful results for interpreting and scaling‐up experiments in clone screening and bioprocess development. Biotechnol. Bioeng. 2009;103: 1118–1128. © 2009 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.     

Quantification of power consumption and oxygen transfer characteristics of a stirred miniature bioreactor for predictive fermentation scale-up

Miniature parallel bioreactors are becoming increasingly important as tools to facilitate rapid bioprocess design. Once the most promising strain and culture conditions have been identified a suitable scale-up basis needs to be established in order that the cell growth rates and product yields achieved in small scale optimization studies are maintained at larger scales. Recently we have reported on the design of a miniature stirred bioreactor system capable of parallel operation [Gill et al. (2008); Biochem Eng J 39:164-176]. In order to enable the predictive scale-up of miniature bioreactor results the current study describes a more detailed investigation of the bioreactor mixing and oxygen mass transfer characteristics and the creation of predictive engineering correlations useful for scale-up studies. A Power number of 3.5 for the miniature turbine impeller was first established based on experimental ungassed power consumption measurements. The variation of the measured gassed to ungassed power ratio, P(g)/P(ug), was then shown to be adequately predicted by existing correlations proposed by Cui et al. [Cui et al. (1996); Chem Eng Sci 51:2631-2636] and Mockel et al. [Mockel et al. (1990); Acta Biotechnol 10:215-224]. A correlation relating the measured oxygen mass transfer coefficient, k(L)a, to the gassed power per unit volume and superficial gas velocity was also established for the miniature bioreactor. Based on these correlations a series of scale-up studies at matched k(L)a (0.06-0.11 s(-1)) and P(g)/V (657-2,960 W m(-3)) were performed for the batch growth of Escherichia coli TOP10 pQR239 using glycerol as a carbon source. Constant k(L)a was shown to be the most reliable basis for predictive scale-up of miniature bioreactor results to conventional laboratory scale. This gave good agreement in both cell growth and oxygen utilization kinetics over the range of k(L)a values investigated. The work described here thus gives further insight into the performance of the miniature bioreactor design and will aid its use as a tool for rapid fermentation process development.     

Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production

Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K _L a and of mixing (via pH measurements) in bioreactors up to 8 m³ at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO₂ has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.     

A comparative study of solid and liquid non‐aqueous phases for the biodegradation of hexane in two‐phase partitioning bioreactors

A comparative study of the performance of solid and liquid non‐aqueous phases (NAPs) to enhance the mass transfer and biodegradation of hexane by Pseudomonas aeruginosa in two‐phase partitioning bioreactors (TPPBs) was undertaken. A preliminary NAP screening was thus carried out among the most common solid and liquid NAPs used in pollutant biodegradation. The polymer Kraton G1657 (solid) and the liquid silicone oils SO20 and SO200 were selected from this screening based on their biocompatibility, resistance to microbial attack, non‐volatility and high affinity for hexane (low partition coefficient: K = C_g/C_NAP, where C_g and C_NAP represent the pollutant concentration in the gas phase and NAP, respectively). Despite the three NAPs exhibited a similar affinity for hexane (K ≈ 0.0058), SO200 and SO20 showed a superior performance to Kraton G1657 in terms of hexane mass transfer and biodegradation enhancement. The enhanced performance of SO200 and SO20 could be explained by both the low interfacial area of this solid polymer (as a result of the large size of commercial beads) and by the interference of water on hexane transfer (observed in this work). When Kraton G1657 (20%) was tested in a TPPB inoculated with P. aeruginosa, steady state elimination capacities (ECs) of 5.6 ± 0.6 g m^?3 h^?1 were achieved. These values were similar to those obtained in the absence of a NAP but lower compared to the ECs recorded in the presence of 20% of SO200 (10.6 ± 0.9 g m^?3 h^?1). Finally, this study showed that the enhancement in the transfer of hexane supported by SO200 was attenuated by limitations in microbial activity, as shown by the fact that the ECs in biotic systems were far lower than the maximum hexane transfer capacity recorded under abiotic conditions. Biotechnol. Bioeng. 2010;106: 731–740. © 2010 Wiley Periodicals, Inc.     

Putting cells under pressure: a simple and efficient way to enhance the productivity of medium-chain-length polyhydroxyalkanoate in processes with Pseudomonas putida KT2440

The success of bioprocess implementation relies on the ability to achieve high volumetric productivities and requires working with high‐cell‐density cultivations. Elevated atmospheric pressure might constitute a promising tool for enhancing the oxygen transfer rate (OTR), the major growth‐limiting factor for such cultivations. However, elevated pressure and its effects on the cellular environment also represent a potential source of stress for bacteria and may have negative effects on product formation. In order to determine whether elevated pressure can be applied for enhancing productivity in the case of medium‐chain‐length polyhydroxyalkanoate (mcl‐PHA) production by Pseudomonas putida KT2440, the impact of a pressure of 7 bar on the cell physiology was assessed. It was established that cell growth was not inhibited by this pressure if dissolved oxygen tension (DOT) and dissolved carbon dioxide tension (DCT) were kept below ～30 and ～90 mg L^?1, respectively. Remarkably, a little increase of mcl‐PHA volumetric productivity was observed under elevated pressure. Furthermore, the effect of DCT, which can reach substantial levels during high‐cell‐density processes run under elevated pressure, was investigated on cell physiology. A negative effect on product formation could be dismissed since no significant reduction of mcl‐PHA content occurred up to a DCT of ～540 mg L^?1. However, specific growth rate exhibited a significant decrease, indicating that successful high‐cell‐density processes under elevated pressure would be restricted to chemostats with low dilution rates and fed‐batches with a small growth rate imposed during the final part. This study revealed that elevated pressure is an adequate and efficient way to enhance OTR and mcl‐PHA productivity. We estimate that the oxygen provided to the culture broth under elevated pressure would be sufficient to triple mcl‐PHA productivity in our chemostat system from 3.4 (at 1 bar) to 11 g L^?1 h^?1 (at 3.2 bar). Biotechnol. Bioeng. 2012; 109:451–461. © 2011 Wiley Periodicals, Inc.     

Kinetics of biodegradation of p-xylene and naphthalene and oxygen transfer in a novel airlift immobilized bioreactor

The scope of this study included the biodegradation performance and the rate of oxygen transfer in a pilot-scale immobilized soil bioreactor system (ISBR) of 10-L working volume. The ISBR was inoculated with an acclimatized population of contaminant degrading microorganisms. Immobilization of microorganisms on a non-woven polyester textile developed the active biofilm, thereby obtaining biodegradation rates of 81 mg/L x h and 40 mg/L x h for p-xylene and naphthalene, respectively. Monod kinetic model was found to be suitable to correlate the experimental data obtained during the course of batch and continuous operations. Oxygen uptake and transfer rates were determined during the batch biodegradation process. The dynamic gassing-out method was used to determine the oxygen uptake rate (OUR) and volumetric oxygen mass transfer, K(L) a. The maximum volumetric OUR of 255 mg O(2)/L x h occurred approximately at 720-722 h after inoculation, when the dry weight of biomass concentration was 0.67 g/L.     

Scale-up from shake flasks to fermenters in batch and continuous mode with Corynebacterium glutamicum on lactic acid based on oxygen transfer and pH

Scale-up from shake flasks to fermenters has been hampered by the lack of knowledge concerning the influence of operating conditions on mass transfer, hydromechanics, and power input. However, in recent years the properties of shake flasks have been described with empirical models. A practical scale-up strategy for everyday use is introduced for the scale-up of aerobic cultures from shake flasks to fermenters in batch and continuous mode. The strategy is based on empirical correlations of the volumetric mass transfer coefficient (k(L) a) and the pH. The accuracy of the empirical k(L) a correlations and the assumptions required to use these correlations for an arbitrary biological medium are discussed. To determine the optimal pH of the culture medium a simple laboratory method based on titration curves of the medium and a mechanistic pH model, which is solely based on the medium composition, is applied. The effectiveness of the scale-up strategy is demonstrated by comparing the behavior of Corynebacterium glutamicum on lactic acid in shake flasks and fermenters in batch and continuous mode. The maximum growth rate (micro(max) = 0.32 h(-1)) and the oxygen substrate coefficient (Y O2 /S= 0.0174 mol/l) of C. glutamicum on lactic acid were equal for shake flask, fermenter, batch, and continuous cultures. The biomass substrate yield was independent of the scale, but was lower in batch cultures (Y(X/S) = 0.36 g/g) than in continuous cultures (Y(X/S) = 0.45 g/g). The experimental data (biomass, respiration, pH) could be described with a simple biological model combined with a mechanistic pH model.     

A simple method to estimate the contribution of biological floc and reactor-solution to mass transfer of oxygen in activated sludge processes

In this study, the mass transfer coefficient of biological floc (K(L)a(bf)) was estimated from the mass transfer coefficient of the mixed-liquor (K(L)a(f)) and the reactor-solution (K(L)a(e)). The biological floc resistance (BFR) and reactor-solution resistance (SR) were defined as the reciprocal of K(L)a(bf) and K(L)a(e), respectively, by applying the concept of serial-resistance originally presented in two-film theory (Lewis and Whitman (1924) Ind Eng Chem 16:1215-1220). The specific biological floc resistance (SBFR) was defined as biological floc resistance per unit biomass concentration. The data indicated that an activated sludge process yielding low BFR/MLR and BFR/SR tended to produce higher oxygen transfer efficiency. Surprisingly, the reactor-solution posed the same level of resistance as clean water in all experiments, except in a 5-day SRT, non-nitrifying, completely mixed activated sludge (CMAS) process run. Furthermore, SBFR successfully represented biological floc and showed a positive correlation to sludge volume index (SVI). In addition, SBFR/SR and oxygen transfer efficiency (OTE(f)) followed an exponential relationship for the complete data set. The method of separating the mixed-liquor into biological floc and reactor-solution improved the understanding of oxygen transfer under process conditions, without resorting to intrusive techniques or direct handling of fragile biological floc.