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     

Monitoring gradient formation in a jet aerated bioreactor

Jet aerated loop reactors (JLRs) provide high mass transfer coefficients (k_La) and can be used for the intensification of mass transfer limited reactions. The jet loop reactor achieves higher k_La values than a stirred tank reactor (STR). The improvement relies on significantly higher local power inputs (～10⁴) than those obtainable with the STR. Operation at high local turnover rates requires efficient macromixing, otherwise reactor inhomogeneities might occur. If sufficient homogenization is not achieved, the selectivity of the reaction and the respective yields are decreased. Therefore, the balance between mixing and mass transfer in jet loop reactors is a critical design aspect. Monitoring the dissolved oxygen levels during the turnover of a steady sodium sulfite feed implied the abundance of gradients in the JLR. Prolonged mixing times at identical power input and aeration rates (～100%) were identified for the JLR in comparison to the STR. The insertion of a draft tube to the JLR led to a more homogenous dissolved oxygen distribution, but unfortunately a reduction of mixing time was not achieved. In case of increased medium viscosities as they may arise in high cell density cultivations, no gradient formation was detected. However, differences in medium viscosity significantly altered the mass transfer and mixing performance of the JLR.     

High density cultivation of plant cells in a new aeration-agitation type fermentor,maxblend fermentor®

The applicability of a new aeration-agitation type fermentor with a grid-paddle type impeller and a spiral-sparger, Maxblend Fermentor® (MBF) for high density cultivation of plant cells, was investigated. The MBF showed a high capacity for oxygen supply and extremely low hydrodynamic stress in aeration and mixing compared with a conventional fermentor (CF). When Oryza sativa cells were cultivated at a k_La of 20 h⁻¹, a high cell density cultivation of about 30 g dry cell weight per liter was accomplished in both fermentors and there were few differences in culture performance between the two. On the contrary, considerable differences were observed when Catharanthus roseus cells, which seemed to be sensitive to physical stress, were cultivated at a k_La of 20 h⁻¹ in both fermentors. The MBF exhibited excellent cell growth characteristics, achieving about 19 g dry cell weight per liter, because of its superior oxygen supply and low hydrodynamic stress in aeration and mixing in highly viscous cultures containing high density cells. In CF only about 9.5 g dry cell weight per liter was achieved because of its high hydrodynamic stress.     

On-line estimation of kLa by an analog computer

A new method was developed for estimating the volumetric oxygen transfer coefficient, k_La, in a fermentor. Various methods were investigated for the on-line estimation of k_La with an analog computer employing a steepest-descent calculation technique. The method by which k_La is estimated (by minimizing the error residue of the model) was found to be very applicable. A method for the simultaneous estimation of the volumetric oxygen transfer coefficient and respiration rate in biological systems is also presented.     

Oxygen transfer in a stirred loop fermentor with dilute polymer solutions

Oxygen transfer in a 0.35 m diameter stirred loop fermentor (a stirred tank with a concentric draft tube) has been studied with water containing a small amount of polymer(polyethylene oxide) as a drag-reducing additive.Power consumption was measured. It was found that the addition of polyethylene oxide causes an increase of power consumption. This is contrary to the results reported in the literature.Volumetric mass transfer coefficients (K _La) were measured. In water the introduction of the draft tube increased the K _La coefficient. The increase in K _La became larger with impeller speed. On the other hand, mass transfer in dilute polymer solutions decreased due to the presence of the draft tube. An empirical correlation has been proposed for the volumetric mass transfer coefficient in stirred loop fermentors. It has a general applicability.List of Symbols a 1/m specific surface area - C constant in Eq. (6) - g m/s² gravitational acceleration - K _L m/s overall liquid-phase mass transfer coefficient - n 1/s impeller speed - P W aerated power input by mechanical agitation - P _g W power input by sparged air - Q m³/min volumetric gas flow rate - U _sg m/s superficial gas velocity - V m³ liquid volume Greek Symbols exponents in Eq. (3) - exponent in Eq. (6) - kg/m³ density     

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.     

Effect of ventilation on the production of acetaldehyde by Zymomonas mobilis

The production of acetaldehyde, a flavoring agent in food, by Zymomonas mobilis was carried out in batch culture. The volatilization rate constant (k_v) of acetaldehyde and the initial volumetric oxygen transfer coefficient (k_La⁰) in an Erlenmeyer flask with a cotton-plug (cotton-flask) and an aerated-flask with a forced-air system (aerated-flask) were measured. The culture environment in the aerated-flask was found to be very different from that in the cotton-flask. Cell growth in a cotton-flask was strongly inhibited, making practical acetaldehyde production in cotton-flask very difficult. On the other hand, acetaldehyde production in the aerated-flask increased while the fermentation time decreased with increases in the air flow rate. The k_v value of acetaldehyde in a jar fermentor was affected mainly by air flow rate. By considering both the oxygen transfer rate and the ventilation effect on the culture, it was possible to scale-up from the aerated-flask to a jar fermentor. In the jar fermentor, production of acetaldehyde and growth inhibition by acetaldehyde were affected mainly by the k_La⁰ and k_v, values, respectively. The overall production of acetaldehyde in the jar fermentor under the optimum k_La⁰ and k_v conditions was 6.64 g/l (Y_p/s: 0.27), which was about 1.5 times higher than the maximum concentration obtained in the aerated-flask.     

Overall volumetric mass transfer coefficient in a modified reversed flow jet loop bioreactor with low density particles

Experiments were conducted using glass beads and low-density particles such as polyurethane and polystyrene which are comparable to bioparticles found in biological applications to evaluate the overall volumetric mass transfer coefficient (K _L a) in a modified reversed flow jet loop bioreactor having the liquid outlet at the top section of the reactor. The influence of the gas and liquid flow rates, draft tube to reactor diameter ratio, solids loading and physical properties of solids onK _L a were studied. TheK _L a was found to increase with the increased gas and liquid flow rates. TheK _L a values were found to be higher in the bubbly flow region i.e., at the lower range of energy dissipation rates. The optimum draft tube to reactor diameter ratio and solids loading with respect to maximumK _L a were found to be 0.4 and 0.9×10^?3 m³ (? _s =0.025) respectively. Dimensionless correlations were presented to predict the experimental values in terms of operational and geometrical variables.     

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     

Saccharomyces cerevisiae fermentations in a pilot scale airlift bioreactor: Comparison of air sparger configurations

Hydrodynamic and oxygen transfer comparisons were made between two ring sparger locations, draft tube and annulus, in a concentric pilot scale airlift reactor with a baker's yeast suspension. Sectional hydrodynamic measurements were made and a mobile DOT probe was used to characterise the oxygen transfer performance through the individual sections of the reactor. The hydrodynamic performance of the reactor was improved by using a draft tube ring sparger rather than the annulus ring sparger. This was due to the influence of the ratio of the cross sectional area of the downcomer and riser (A _D/A_R) in conjunction with the effect of liquid velocity and a parameter,C ₀, describing the distribution of the liquid velocity and gas holdup across the riser on the bubble coalescence rates. The mixing performance of the reactor was dominated by the frequency of the passage of the broth through the end sections of the reactor. An optimum liquid height above the draft tube, for liquid mixing was demonstrated, above which no further improvement in mixing occurred. The liquid velocity and degree of gas entrainment showed little dependency on top section size for both sparger configurations. Extreme dissolved oxygen heterogeneity was demonstrated around the vessel with both sparger configurations and was shown to be detrimental to the oxygen uptake rate of the baker's yeast. Dissolved oxygen tensions below 1% air saturation occurred along the length of the riser and then rose in the downcomer. The greater oxygen transfer rate in the downcomer than in the riser was caused by the combined effects of a larger slip velocity in the downcomer which enhancedk _La and gas residence time, high downcomer gas holdup, and the change in bubble size distribution between the riser and downcomer. The position of greatest oxygen transfer rate in the downcomer was shown to be affected by the reactor from the influence on downcomer liquid linear velocity. UCL is the Biotechnology and Biological Sciences Research Council sponsored Advanced Centre for Biochemical Engineering and the Council's support is greatly acknowledged.     

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.     

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.     

Xylitol Production from D‐Xylose at Different Oxygen Transfer Coefficients in a Batch Bioreactor

Conversion of D‐xylose to xylitol by Candida boidinii NRRL Y‐17213 was studied under anaerobic and oxygen limited conditions by varying the oxygen transfer coefficient k_La. Shake flask experiments were used to provide the preliminary information required to perform experiments in a bioreactor. The yeast did not grow under fully anaerobic conditions, but anaerobic formations of xylitol, ethanol, ribitol, and glycerol were observed as well as D‐xylose assimilation of 11 %. In shake flasks, with an initial D‐xylose concentration of 50 g/L, an increase in k_La from 8 to 46 h^–1 resulted in a faster growth, higher rate of substrate uptake and lower yields of products. The highest xylitol productivity (0.052 g/L h) was attained at k_La = 8 h^–1. At k_La = 46 h^–1, 98.6 % of D‐xylose was consumed and mainly converted to biomass. Using 130 g/L D‐xylose, k_La was varied in the fermenter from 26 to 78 h^–1. The percentage of consumed D‐xylose increased from 31 % at k_La = 26 h^–1 to 93–94 % at all other aeration levels. Biomass yield increased with k_La, whereas ethanol, ribitol, and glycerol yields exhibited an opposite dependence on the oxygenation level. The most favorable oxygen transfer coefficient for xylitol formation, in the fermenter, was k_La = 47 h^–1 when its concentration (57.5 g/L) surpassed ethanol accumulation by 3.6‐fold, and the glycerol plus ribitol by 10‐fold. Concurrently, xylitol yield and productivity reached 0.45 g/g and 0.26 g/L h, respectively. The volumetric xylitol productivity was affected more by changes in the aeration than the corresponding yield.     

Mixing performance of air-lift fermenters against working volume and draft tube dimensions

Mixing characteristics of a laboratory scale internal loop air-lift fermenter has been investigated. The effects of different draft tube dimensions and positions as well as varying levels of liquid height over the draft tube, on mixing time were determined. The results indicate the existance of an optimum liquid height and thus liquid volume with respect to mixing performance especially for the taller draft tubes.List of Symbols A mm distance between draft tube and reactor base - A _D mm² area of the downcomer region - A _R mm² area of the riser region - B mm width of annulus - D _d mm draft tube diameter - D _t mm fermenter diameter - H _d mm draft tube height - H _l mm liquid height in the fermenter - H _t mm fermenter height - V _d m³ draft tube volume - V _t m³ fermenter volume - D _d /D _l - _B H _d /H _l - _F H _l /D _t      

Desorption of carbon dioxide from fermentation broth

Rates of CO₂ desorption from fermentation broths under actual operating conditions were determined by measuring the CO₂ partial pressure in the exit gas. The concentrations of CO₂ physically dissolved in the broths were measured by the so-called tubing method. Values of k_La for CO₂ desorption calculated from these values agreed well with the k_La values for oxygen absorption corrected for the difference in gas diffusivities. The dissolved CO₂ concentration in the broth, which seems to bean important operating parameter, can easily be estimated from the CO₂ partial pressure in the exit gas, a more easily measurable quantity, if the k_La value is known. For a given value of k_La, assumption of perfect mixing or plug flow in the gas phase made little difference in the calculated values of the dissolved CO₂ concentration, indicating that the gas phase was probably in between perfect mixing and plug flow. In industrial fermentors, the CO₂ partial pressure in the exit gas can practically be assumed to be in equilibrium with the dissolved CO₂ concentration.     

Pilot scale production of poly(3-hydroxybutyrate-co-3-hydroxy-valerate) by fed-batch culture of recombinantEscherichia coli

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB/V)], by fed-batch culture of recombinantEscherichia coli harboring a plasmid containing theAlcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes, was examined in two pilot-scale fermentors with air supply only. In a 30 L fermentor having aK _La value of 0.11 s⁻¹, the final P(3HB/V) concentration and the P(3HB/V) content obtained were 29.6 g/L and 70.1 wt%, respectively, giving a productivity of 1.37 g P(3HB/V)/L-h. In a 300 L fermentor having aK _La of 0.03 s⁻¹, the P(3HB/V) concentration and the P(3HB/V) content were 20.4 g/L and 69 wt%, respectively, giving a productivity of 1.06 g P(3HB/V)/L-h. These results suggest that economical production of P(3HB/V) is possible by fed-batch culture of recombinantE. coli in a large-scale fermentor having lowK _La value.     

A case study in converting disposable process scouting devices into disposable bioreactors as a future bioprocessing tool

In this study, we perform mass transfer characterization (k_La) on a novel mechanically driven/stirred Process Scouting Device, PSD, (SuperSpinner D 1000®, SSD) and demonstrate that this novel device can be viewed as disposable bioreactor. Using patch‐based optical sensors, we were able to monitor critical cell culture environmental conditions such as dissolved oxygen (DO) and pH in SSD for comparison to a 1 L standard spinner (SS) flask. We also coupled these mass transfer studies with mixing time studies where we observed relative high mixing times (5.2 min) that are typically observed in production scale bioreactors. Decreasing the mixing time 3.5‐fold resulted in 30% increase in k_La (from 2.3 to 3.0 h^?1) and minimum DO level increased from 0% to 20% for our model hybridoma cell line. Finally, maximum viable cell density and protein titer stayed within ±20% of historical data, from our standard 5 L stirred bioreactor (Biostat®) operated under active DO control. Biotechnol. Bioeng. 2012; 109: 2790–2797. © 2012 Wiley Periodicals, Inc.     

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

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

Mixing time in a down-flow jet loop bioreactor

Mixing time was determined in a down-flow jet loop bioreactor with Newtonian and non-Newtonian fluids. It was observed that the mixing time decreased with an increase in linear liquid velocity, superficial gas velocity, draft tube to column diameter ratio, nozzle diameter and shear thinning of media. The optimum draft tube to column diameter ratio was found to be about 0.44. Correlations were presented for prediction of mixing time.List of Symbols A m² cross sectional area of the column - C kmol/m³ local tracer concentration - A _D m² flow area,A _D =/4 (D _Z ² -D _TO ² ) - D m column diameter - D _E m draft tube diameter - D _TO m outside diameter of the air tube - D _TFL m equivalent flow diameter,D _TFL =(D _Z ² -D _TO ² )^0.5 - D _z m nozzle diameter - g m/s² gravitational acceleration - h % inhomogeneity - H m height of the column - H _B m distance between the lower edge of the draft tube and the impact plate - H _T m distance between the upper edge of the draft tube and the liquid nozzle - K Pa.sⁿ consistency index in power-law model - L _E m length of the draft tube - n flow index in the power-law model - Re _j jet Reynolds number,Re _j =(D _TFL×w₁×_L)/ _eff - t _M s mixing time - t _sg m/s superficial gas velocity based onA - W _l m/s linear liquid velocity based onD _D Greek Letters N/m² shear stress - s shear rate - kg/m³ density of liquid - N/m surface tension of the liquid - Pa.sⁿ viscosity of liquid Indices X concentration at infinite time maximum value of tracer concentration - eff effective - L Liquid - obs observed - pred Predicted