Volumetric Oxygen Transfer Coefficient Research Articles

New milliliter‐scale stirred tank bioreactors for the cultivation of mycelium forming microorganisms

A novel milliliter-scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms on a 10 milliliter-scale. A newly designed one-sided paddle impeller is driven magnetically and rotates freely on an axis in an unbaffled reaction vessel made of polystyrene. A rotating lamella is formed which spreads out along the reactor wall. Thus an enhanced surface-to-volume ratio of the liquid phase is generated where oxygen is introduced via surface aeration. Volumetric oxygen transfer coefficients (k(L)a) > 0.15 s(-1) were measured. The fast moving liquid lamella efficiently prevents wall growth and foaming. Mean power consumption and maximum local energy dissipation were measured as function of operating conditions in the milliliter-scale stirred tank bioreactor (V = 10 mL) and compared to a standard laboratory-scale stirred tank bioreactor with six-bladed Rushton turbines (V = 2,000 mL). Mean power consumption increases with increasing impeller speed and shows the same characteristics and values on both scales. The maximum local energy dissipation of the milliliter-scale stirred tank bioreactor was reduced compared to the laboratory-scale at the same mean volumetric power input. Hence the milliliter impeller distributes power more uniformly in the reaction medium. Based on these data a reliable and robust scale-up of fermentation processes is possible. This was demonstrated with the cultivation of the actinomycete Streptomyces tendae on both scales. It was shown that the process performances were equivalent with regard to biomass concentration, mannitol consumption and production of the pharmaceutical relevant fungicide nikkomycin Z up to a process time of 120 h. A high parallel reproducibility was observed on the milliliter-scale (standard deviation < 8%) with up to 48 stirred tank bioreactors operated in a magnetic inductive drive. Rheological behavior of the culture broth was measured and showed a highly viscous shear-thinning non-Newtonian behavior. The newly developed one-sided paddle impellers operated in unbaffled reactors on a 10 milliliter-scale with a magnetic inductive drive for up to 48 parallel bioreactors allows for the first time the parallel bioprocess development with mycelium forming microorganisms. This is especially important since these kinds of cultivations normally exhibit process times of 100 h and more. Thus the operation of parallel stirred tank reactors will have the potential to reduce process development times drastically.

Construction of recombinant Escherichia coli for enhanced bioconversion of colchicine into 3-demethylated colchicine at 70l bioreactor level

Abstract A recombinant strain of Escherichia coli with CYP102A1 gene was developed for the demethylation of colchicine into their derivatives. The CYP102A1 gene responsible for demethylation was isolated from Bacillus megaterium ACBT03 and amplified using suitable primers. The amplified product was cloned into pET28a+ expression vector using host E. coli BL21(DE3) cells. The CYP3A4 (product of CYP102A1 gene) protein expression and other parameters like substrate toxicity, product toxicity and enzyme activity were optimized in shake flasks; and further scaled-up to 5 l bioreactor with 3 l working volume. In 5 l bioreactor, dissolved oxygen (DO) was optimized for maximum specific growth and enhanced 3-demethylated colchicine (3-DMC) production. The optimized conditions from shake flasks were scaled-up to 70 l bioreactor and resulted into ∼80% conversion of 20 mM colchicine in 48 h with a volumetric productivity of 6.62 mg l−1 h−1. Scale-up factors were measured as volumetric oxygen transfer coefficient (kLa) i.e., 56 h−1 and impeller tip velocity (Vtip) i.e., 7.065 m s−1, respectively. The kinetic parameters Km, kcat, and kcat/Km of the CYP3A4 enzyme using colchicine as the substrate were determined to be 271 ± 30 μM, 8533 ± 25 min−1, and 31.49 μM min−1, respectively, when IPTG induced recombinant E. coli culture was used.

A shaken disposable bioreactor system for controlled insect cell cultivations at milliliter‐scale

AbstractWhile wave‐mixed and stirred bag bioreactors are common devices for rapid, safe insect cell culture‐based production at liter‐scale, orbitally shaken disposable flasks are mainly used for screening studies at milliliter‐scale. In contrast to the two aforementioned bag bioreactor types, which can be operated with standard or disposable sensors, shaker flasks have not been instrumented until recently. The combination of 250 mL disposable shake flasks with PreSens's Shake Flask Reader enables both pH and dissolved oxygen to be measured, as well as allowing characterization of oxygen mass transfer. Volumetric oxygen transfer coefficients (kLa‐values) for PreSens 250 mL disposable shake flasks, which were determined for the first time in insect cell culture medium at varying culture volumes and shaker frequencies, ranged between 4.4 and 37.9/h. Moreover, it was demonstrated that online monitoring of dissolved oxygen in shake flasks is relevant for limitation‐free growth of insect cells up to high cell densities in batch mode (1.6×107 cells/mL) and for the efficient expression of an intracellular model protein.

Gas Hold-Up and Mass Transfer in Three Geometrically Similar Internal Loop Airlift Reactors Using Newtonian Fluids

Global gas hold-up and volumetric oxygen transfer coefficient (kLa) were evaluated in three internal-loop airlift reactors (ALR) with different working volumes (2, 5, and 10 dm3) and similar geometric configuration utilizing eight Newtonian fluids. Superficial gas velocity in the riser (UGR) was ranged from 0 to 0.112 ms-1. The effects of the superficial gas velocity (UGR) and liquid viscosity had contrary effects on kLa. However, they presented the same order of magnitude showing that the viscosity effect on the oxygen mass transfer cannot be neglected. Two correlations based on dimensional analysis relating global gas hold-up and kLa to the geometric parameters, physical properties of the fluids, and operational conditions were proposed. Very good fittings between calculated and experimental data of and kLa were obtained. The influence of the internal diameter of the reactor on kLa was considered positive showing that in large-scale reactors an appropriate oxygen transfer can be reached under smaller aeration conditions, due to higher internal diameter.

Scale up and Application of Biosurfactant from Bacillus subtilis in Enhanced Oil Recovery

There is a lack of fundamental knowledge about the scale up of biosurfactant production. In order to develop suitable technology of commercialization, carrying out tests in shake flasks and bioreactors was essential. A reactor with integrated foam collector was designed for biosurfactant production using Bacillus subtilis isolated from agricultural soil. The yield of biosurfactant on biomass (Y(p/x)), biosurfactant on sucrose (Y(p/s)), and the volumetric production rate (Y) for shake flask were obtained about 0.45 g g(-1), 0.18 g g(-1), and 0.03 g l(-1) h(-1), respectively. The best condition for bioreactor was 300 rpm and 1.5 vvm, giving Y(x/s), Y(p/x), Y(p/s), and Y of 0.42 g g(-1), 0.595 g g(-1), 0.25 g g(-1), and 0.057 g l(-1) h(-1), respectively. The biosurfactant maximum production, 2.5 g l(-1), was reached in 44 h of growth, which was 28% better than the shake flask. The obtained volumetric oxygen transfer coefficient (K(L)a) values at optimum conditions in the shake flask and the bioreactor were found to be around 0.01 and 0.0117 s(-1), respectively. Comparison of K(L)a values at optimum conditions shows that biosurfactant production scaling up from shake flask to bioreactor can be done with K(L) a as scale up criterion very accurately. Nearly 8% of original oil in place was recovered using this biosurfactant after water flooding in the sand pack.

Effect of silicone oil fraction and stirring rate on methane degradation in a stirred tank reactor

AbstractBACKGROUND: Methane is an important greenhouse gas and its biological oxidation constitutes a cost‐effective alternative for low concentration emissions. Nevertheless, due to its low aqueous solubility (similar to oxygen), gas mass transfer is often the limiting step. Two‐phase partition bioreactors (TPPBs), have been proposed to increase the biodegradation of poorly soluble compounds. In this work, the effect of stirring rate and silicone oil fraction on abiotic volumetric oxygen transfer coefficient kLa and methane elimination capacity (EC) by a methanotrophic consortium in a stirred tank was studied.RESULTS: In abiotic conditions, the kLa increased when increasing both factors. Nevertheless, a negative effect on kLa was observed with silicone oil at the highest stirring rate (800 rpm). During methane biodegradation experiments, EC also improved when increasing both factors, but contrary to abiotic kLa, it was not affected at the highest stirring rate. While stirring rate was the dominant variable determining methane biodegradation, the effect of oil fraction was only significant above 200 rpm. A maximum EC improvement of 700% was observed when increasing stirring rate from 200 to 800 rpm with 10% of silicone oil.CONCLUSIONS: Increasing both stirring rate and oil fraction enhanced EC. The results were different from those observed with kLa suggesting that kLa is not sufficient to describe properly the mass transfer in TPPB because other factors (interfacial contact between the phases and physicochemical properties of organic phase) should be considered. Copyright © 2010 Society of Chemical Industry

Interfacial gas–liquid transfer area in alkane–aqueous dispersions and its impact on the overall volumetric oxygen transfer coefficient

K L a and gas–liquid interfacial area per unit volume were quantified in 2.5–20% n-C 10–13 aqueous dispersions agitated at 600–1200 rpm. The interfacial area was quantified using high speed photography and image analysis. Interfacial area correlated strongly with K L a over all agitation rates and alkane concentrations, suggesting that the interfacial area was the major factor defining K L a in these dispersions. The bubble diameter and gas hold up were identified as key parameters defining an optimal K L a at 5% alkane at agitation rates of 800–1200 rpm, through their impact on the interfacial area. Decreased bubble diameter and increased gas hold up with alkane addition from 2.5% to 5% resulted in increased interfacial area. However, a decreased gas hold up on alkane addition above 5% resulted in a correspondingly decreased interfacial area, despite the continued decline in bubble diameter. The decrease in bubble diameter and the decrease in gas hold up have in part been attributed to the reduction of surface tension from 26.0 to 17.7 and the doubling of viscosity to 2.0 mN/m respectively over the range of alkane concentrations examined.

Effects of Oxygen Supply on Growth and Carotenoids Accumulation by Xanthophyllomyces dendrorhous

The effects of oxygen supply on growth and carotenoids accumulation by Xanthophyllomyces dendrorhous were studied. Initial volumetric oxygen transfer coefficients (K(L)a) within the range 21.5-148.5 h(-1) had significant effects on growth and carotenoids accumulation, and an increase of the initial K(L)a value led to higher carotenoids, astaxanthin and biomass yields by X. dendrorhous. At an initial K(L)a value of 148.5 h(-1), a maximal cell concentration of 19.37 g 1(-1) and optimal carotenoids and astaxanthin productions of 18.1 and 14.5 mg 1(-1) were obtained, as well as a maximal astaxanthin content of 0.8 mg g DCW(-1), respectively. A higher oxygen supply was advantageous to astaxanthin biosynthesis and the ratio of astaxanthin in the total carotenoids. An increasing initial K(L)a value gave stronger fluorescence intensities by X. dendrorhous, resulting in the maximal intensity of fluorescence at the K(L)a value 148.5 h(-1). The cell growth of X. dendrorhous was significantly inhibited when dissolved oxygen tension (DOT) was controlled at approximately 20% air saturation, which was due to the oxygen limitation in broth. The astaxanthin yield and content at approximately 50% DOT were higher than those at approximately 20% DOT.

Development of a three-phase fluidized bed reactor with enhanced oxygen transfer

A three-phase fluidized bed reactor (TFBR) was developed in this study with the objective to achieve high rates of oxygen transfer from the gas to the liquid phase in the presence of fluidized solid particles. With 2.9 m height, 0.605 m diameter, and a short residence time of 8 h, the TFBR is particularly suitable for industrial applications such as aerobic biodegradation of high-strength wastewaters including refractory compounds. Experiments with tap water and air show that the TFBR enables complete fluidization. With the water and air superficial velocities in the respective ranges of 0.005–0.203 and 0.8–2.0 cm/s, the volumetric oxygen transfer coefficient is 2.3 × 10 −2 s −1, which is higher than that obtained in similar experimental studies on oxygen transfer carried out in the past. These results suggest that the developed TFBR could be very effective in industrial applications where short hydraulic time and high gas-to-liquid mass transfer rates are desirable.

Dynamic measurement of the volumetric oxygen transfer coefficient in fermentation systems

A rapid and internally consistent technique has been developed to measure the volumetric oxygen transfer coefficient, k L a, in fermentation systems. The method consists of tracing the dissolved O 2 concentration of the fermentation broth during a short interruption of the aeration. The O 2 concentration trace thus obtained can be analyzed to determine the values of k L a. Additional experiments on prolonged O 2 starvation, carried out to find the limitation of the technique, suggest that O 2 uptake rate will vary if a prolonged (2-10 min.) O 2 starvation occurs.

Enhancement of Red Pigment Production by Monascus purpureus FTC 5391 through Retrofitting of Helical Ribbon Impeller in Stirred-Tank Fermenter

The possibility of using a novel proximity type half-pitched, double-flight helical ribbon impeller (HRI) for the improvement of red pigment production by Monascus purpureus FTC5391 was investigated in a 2-L stirred-tank fermenter (STF). For comparison, the fermentations were also carried out in STF equipped with the industrial standard six-bladed Rushton turbines (RT). The selection of aeration and agitation strategies in STF were based on possible attainable oxygen transfer at the start of fermentation using derived empirical power law model to predict the initial volumetric oxygen transfer coefficient (kLa). The final pigment concentration, yield, and productivity obtained in optimized fermentation employing a single HRI agitated at 250 rpm and air flow rate of 2.25 L/min (1.5 vvm) was 24.36 UA500, 0.47 UA500/g glucose, and 0.20 UA500/h, respectively. These values were about 76%, 78%, and 96% higher than those obtained in STF fitted with RT, respectively. In terms of impeller power consumption, the HRI system was favorable for batch cultivation of M. purpureus as pigment yield per energy invested was 2.5 times higher than the RT.

Favorable effect of very low initial KLa value on xylitol production from xylose by a self-isolated strain of Pichia guilliermondii

Xylitol production from xylose by a self-isolated furfural and 5-hydroxymethyl furfural assimilating Pichia guilliermondii was studied under oxygen limitation. An extremely low initial volumetric oxygen transfer coefficient (0.075 h(-1)) was found most favorable to the xylitol production with yield of 0.61 g g(-1). Related enzymes activities were also investigated and discussed.

Measurement of the overall volumetric oxygen transfer coefficient in alkane‐aqueous dispersions

AbstractBACKGROUND: A reliable procedure for the determination of the volumetric oxygen transfer coefficient (KLa) in hydrocarbon–aqueous dispersions is critical to the assessment of oxygen transfer in hydrocarbon‐based bioprocesses. The pressure step procedure (PSP) and gassing out procedure (GOP) for KLa determination were evaluated in n‐C10–13 alkane–aqueous dispersions over agitation rates (600 to 1200 rpm) and alkane concentrations (up to 20%) typical of hydrocarbon‐based bioprocesses.RESULTS: KLa from the procedures diverged progressively as agitation increased, with KLa underprediction attenuated at high alkane. At 1200 rpm and 5% alkane, KLa from the GOP was underpredicted by 49%. Compared with KLa from the PSP, KLa from the PSP without nitrogen transport was the same, but KLa from the PSP without response lag (PSP‐lag) was underpredicted by approximately 50% at 1200 rpm. Moreover, KLa from the PSP‐lag was lower than KLa from the GOP, except at 1200 rpm and alkane concentration up to 5%.CONCLUSION: The PSP was superior to the GOP in alkane–aqueous dispersions, particularly at extreme agitation and low alkane concentrations. This was attributable to the probe response lag and the non‐uniformity of gas composition inherent in the GOP and independent of the rate of coincident nitrogen transport. Copyright © 2009 Society of Chemical Industry

Maximization of β-Galactosidase Production: A Simultaneous Investigation of Agitation and Aeration Effects

In this work, the agitation and aeration effects in the maximization of the beta-galactosidase production from Kluyveromyces marxianus CCT 7082 were investigated simultaneously, in relation to the volumetric enzyme activity and the productivity, as well as the analysis of the lactose consumption and production of glucose, and galactose of this process. Agitation and aeration effects were studied in a 2 L batch stirred reactor. A central composite design (2(2) trials plus three central points) was carried out. Agitation speed varied from 200 to 500 rpm and aeration rate from 0.5 to 1.5 vvm. It has been shown in this study that the volumetric enzyme production was strongly influenced by mixing conditions, while aeration was shown to be less significant. Linear models for activity and productivity due to agitation and aeration were obtained. The favorable condition was 500 rpm and 1.5 vvm, which lead to the best production of 17 U mL(-1) for enzymatic activity, 1.2 U mL(-1) h(-1) for productivity in 14 h of process, a cellular concentration of 11 mg mL(-1), and a 167.2 h(-1) volumetric oxygen transfer coefficient.

Gas‐Liquid Mass Transfer in Fibrous Bed Reactor with Counter‐Current Liquid Recycle

AbstractIn this work, the gas‐liquid mass transfer in a lab‐scale fibrous bed reactor with liquid recycle was studied. The volumetric gas‐liquid mass transfer coefficient, kLa, is determined over a range of the superficial liquid velocity (0.0042–0.0126 m.s–1), gas velocity (0.006–0.021 m.s–1), surface tension (35–72 mN/m), and viscosity (1–6 mPa.s). Increasing fluid velocities and viscosity, and decreasing interfacial tension, the volumetric oxygen transfer coefficient increased. In contrast to the case of co‐current flow, the effect of gas superficial velocity was found to be more significant than the liquid superficial velocity. This behavior is explained by variation of the coalescing gas fraction and the reduction in bubble size. A correlation for kLa is proposed. The predicted values deviate within ± 15 % from the experimental values, thus, implying that the equation can be used to predict gas‐liquid mass transfer rates in fibrous bed recycle bioreactors.

Modeling of Oxygen Transfer Correlations for Stirred Tank Bioreactor Agitated with Atypical Helical Ribbon Impeller

Problem Statement: Mixing filamentous fungi entails delicate balance between promoting high volumetric oxygen transfer coefficient (kLa) while keeping low hydrodynamic stresses in the microenvironment of cultures. This study examined the oxygen transfer capability of a prototype low shear helical impeller as a potential replacement for the standard Rushton turbine. Approach: The dependence of kLa upon specific power uptake, superficial gas velocity and apparent viscosity were examined under coalescent, non-coalescent and viscous pseudoplastic fluids scenario using dynamic gassing out technique. Subsequently, collected data were treated under historical data design of response surface methodology. Results: Three empirical power law kLa correlations were developed for each fluid. Correlation comparison with literature models for single turbine agitation suggest higher kLa augmentation within 1.5-3.6 folds for helical impeller in distilled water and as high as 78% improvement in electrolytes fluid. However impeller performance is comparatively 10% lower against theoretical kLa of triple turbines arrangement for power uptake between 0-1600 W m-3. In case of carboxymethylcellulose, better oxygen transfer is expected for design with higher proportionality of impeller-to-tank internal diameter. Conclusions: Helical impeller performance as gas-liquid contactor is comparable to single and triple Rushtons turbines, with superficial gas velocity proved to be more influential than power uptake in non-viscous liquids and vice versa in viscous pseudoplastic fluids.

Hydrodynamic and oxygen mass transfer studies in a three-phase (air–water–ionic liquid) stirred tank bioreactor

Recently, room temperature ionic liquids have been investigated as organic solvent replacements in multiphase partitioning bioreactors. They could prove particularly useful for oxidative bioconversions due to their non-volatile and non-flammable nature. In order to promote high mass transfer rates stirred tank bioreactors are normally used. However, for multiphase systems, particularly those featuring ionic liquids, there is little hydrodynamic information available to underpin bioreactor design and operation. The aim of this work was to study the hydrodynamic and oxygen mass transfer characteristics of a three-phase (gas–water–ionic liquid) stirred tank partitioning bioreactor. A central composite experimental design was used to investigate the interaction of three operational factors, agitation (400, 700, 1000 rpm) and aeration rates (0.5, 0.75, 1.0 vvm), and ionic liquid volume fraction (5, 12.5, 20%), on two response variables, the Sauter mean droplet diameter ( d 32) and the volumetric oxygen transfer coefficient ( k L a). Three different ionic liquids with a range of physicochemical properties were also investigated. The experimental data obtained were fitted to empirical correlations relating d 32 and k L a values to the measured gassed power input and other bioreactor operating parameters. In all cases the measured values of k L a (and in most cases d 32 values) decreased as ionic liquid volume fraction increased as is the case when using conventional organic solvents. It was shown that different physicochemical properties of the ionic liquid tested gave important differences in the drop size behaviour as a function of agitation conditions. Both of the correlations established for describing the variation of k L a and d 32 with bioreactor operating conditions gave reliable predictions of the measured values. These correlations will be useful for ongoing studies on the design and operation of partitioning bioreactors for oxidative bioconversions.

Estimation of dissolved carbon dioxide stripping in a large bioreactor using model medium

Dissolved carbon dioxide (dCO(2)) accumulation is one of the most serious problems in the scale-up of industrial cell culture. To predict the effects of dCO(2) stripping in different culture conditions and at different scales, we examined a method of estimation of dCO(2) stripping using a model medium. The operational parameters (e.g., sparging and agitation rate) and the size of the bioreactor (working volume: 80 L, 500 L, 2000 L; aspect ratio: 1.0 approximately 1.6) were varied, and the model medium was prepared by adjusting pH, density, viscosity, surface tension, and buffer conditions. dCO(2) stripping efficiency was evaluated using the index k(L)a(CO)((2)), which was defined in accordance with the volumetric oxygen transfer coefficient k(L)a. The model medium exhibited dCO(2) stripping behavior similar to real culture medium in all experimental conditions tested. It is expected that the use of the model medium to estimate dCO(2) stripping in real cultures will be valuable for determining the culture conditions in bioreactors in scale-up.

Culture scale-up studies as seen from the viewpoint of oxygen supply and dissolved carbon dioxide stripping

Oxygen supply and dissolved carbon dioxide (dCO(2)) stripping are two of the most important control parameters in cell culture. In this study, we investigated the effect of scale-up on the volumetric gas transfer coefficient with bioreactors of different sizes (working volume: 80 L, 500 L, 2000 L, and 10,000 L; aspect ratio: 1.0-1.6). Sparging air into water increased the volumetric oxygen transfer coefficient (k(L)a), an index of oxygen supply efficiency, by scale-up roughly in proportion to the depth of the water. A corresponding increase in k(L)a was found in a real cell culture of Chinese hamster ovary cells. dCO(2) stripping efficiency was evaluated in water tests using changes in k(L)a(co2), an index defined in relation to k(L)a. k(L)a(co2) increased following surface aeration, but the rate of increase was reduced by scale-up, which was attributed to a decrease in the liquid surface-to-volume ratio. A similar decrease in efficiency was observed in a 2000 L bioreactor by increasing the liquid volume at constant liquid surface area. The observed scale-up effects are discussed based on a simple theoretical consideration.

Optimization of production of caffeine demethylase by Pseudomonas sp. in a bioreactor

The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight(-1) h(-1) was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h(-1), indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors.