A kLa identification technique for a dynamic model of the coupled system: Air-lift fermenter — oxygen probes

The time delay of oxygen probe response to the signal from a fermenter makes identification of the volumetric oxygen transfer coefficient k_La by the dynamic method more complicated. A coupled model involving the transient-state oxygen balance of the fermenter together with the dynamic model of the oxygen probe must be then formulated, solved and identified. In this paper two simple models of air-lift loop fermenters have been proposed and a coupled mathematical model of the fermenter – oxygen probe system has been developed. The identification procedure was used to estimate k_La values in the fermenter with internal circulation flow on the basis of experimental measurements. A comparison of evaluated and experimental indications of the probes placed at various heights of the column proves that the model presented gives a possibility of the first-step approximation of k_La in loop fermenters.     

Microcalorimetric investigations of the metabolism of yeasts. Growth in chemostat cultures on glucose

Summary As part of a project on the production of penicillin, the penicillin production of two strains of Penicillium chrysogenum which have a different penicillin productivity was investigated in bubble column bioreactors and for comparison in stirred fermenters. The main interest of this study were the complicated interrelations between the stirrer speed, the stirrer type, the shear stress, the morphology of the mycelium and broth viscosity as well as the effect of the oxygen transfer behavior on antibiotic productivity.Stirred tank reactors with different turbine stirrers as well as with a draught tube and propeller were employed.The main variable investigated was the stirrer speed. At low stirrer speeds, gas dispersion is inadequate and the insufficient oxygen transfer rate is a limiting factor. At higher stirrer speeds, the oxygen supply of pulpy mycelia is improved and more cell mass is formed. This result is the same for both strains in all three reactors.If the oxygen partial pressure is near the lower cirtical value, a high percentage of the carbon source is converted into penicillin but the penicillin productivity is low due to a low percentage of penicillin producing cells. At oxygen partial pressures just above 8% saturation, the absolute penicillin productivity is maximal. At higher stirrer speeds and dissolved oxygen concentrations the penicillin production phase is shorter, cell growth is higher and a higher percentage of the carbon source is converted into CO₂.In reactors with a draught tube and propeller, a lower productivity is attained than in those with turbine stirrers.The behavior of the two strains is fairly similar. The higher producing strain, however, has a more distinct separation between its periods of growth and production than does the low producing one. At high stirrer speeds the increase in the cell growth rate is less significant and the substrate yield coefficients are higher for the high producing strain than for the low producing one.Symbols C Dissolved oxygen concentration (mg l^–1) - C^* C at saturation (mg l^–1) - k_La Volumetric mass transfer coefficient (h^–1) - OTR Oxygen transfer rate (mg l^–1 h^–1) - OUR Oxygen uptake rate (mg l^–1 h^–1) - rpm Impeller speed (min^–1) - X (Dry) biomass concentration (g kg^–1) - V_g Volumetric gas flow rate (Nl min^–1) - CMC Carboxymethyl cellulose     

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.     

Decolourization of anaerobically digested and polyaluminium chloride treated distillery spentwash in a fungal stirred tank aerobic reactor

Decolourization of anaerobically digested and polyaluminium chloride treated distillery spentwash was studied in a fungal stirred tank aerobic reactor without dilution of wastewater. Aspergillus niger isolate IITB-V8 was used as the fungal inoculum. The main objectives of the study were to optimize the stirrer speed for achieving maximum decolourization and to determine the kinetic parameters. A mathematical model was developed to describe the batch culture kinetics. Volumetric oxygen transfer coefficient (k _L a) was obtained using dynamic method. The maximum specific growth rate and growth yield of fungus were determined using Logistic equation and using Luedeking–Piret equation. 150 rpm was found to be optimum stirrer speed for overall decolourization of 87%. At the optimum stirrer speed, volumetric oxygen transfer coefficient (k _L a) was 0.4957 min⁻¹ and the maximum specific growth rate of fungus was 0.224 h⁻¹. The values of yield coefficient (Y _x/s) and maintenance coefficient (m _s) were found to be 0.48 g cells (g substrate)⁻¹ and 0.015 g substrate (g cells)⁻¹ h⁻¹.     

Effect of stirrer speed,aeration rate and cell mass concentration on volumetric oxygen transfer coefficients (KLa) in the cultivation of Curvularia lunata in a batch reactor

Summary Curvularia lunata was grown in a stirred and aerated reactor for the production of extracellular rifamycin oxidase. Volumetric oxygen transfer coefficients (K_La) were measured for various stirrer speeds, rates of aeration and cell mass concentrations in the reactor. Stirrer speed and aeration rate were optimized and it was found that stirrer speeds of 400–500 rpm and aeration rates of 0.75–1 vvm were optimum for the maximum amount of enzyme production. It was noticed that the increase in cell mass decreased the oxygen transfer coefficient. It was also noticed that the organism formed pellets rather than mycelia when grown on glucose and with an increase in the concentration of glucose in the reactor, there was heavy pellet formation.     

Oxygen transfer measurements during yeast fermentations in a pilot scale airlift fermenter

Measurements of oxygen transfer were made during cultivation of the yeast Saccharomyces cerevisiae in a 90–250 litre working volume concentric tube airlift fermenter. Results demonstrated that the rate of oxygen transfer varies with position in the fermenter, being higher in the riser and top-section than in the downcomer and lowest near the base of the fermenter. The time for liquid circulation was generally smaller than the time constant for oxygen transfer (1/k_La) indicating that the rate of oxygen transfer was slow compared to the rate of liquid movement. Measured dissolved oxygen concentrations therefore did not represent the equilibrium arising from the balance between the rates of oxygen transfer and oxygen depletion. Hence measuredk _L a values were not representative of local oxygen transfer conditions but instead were indicators of the rate of mass transfer the liquid flow had encountered prior to reaching the point of measurement. Generally the individual rates of oxygen transfer in the vessel were found to increase with increasing vessel height.     

Effect of cycling dissolved oxygen concentrations on product formation in penicillin fermentations

Summary Limitations in mass and momentum transfer coupled with high hydrostatic pressures create significant spatial variations in dissolved gas concentrations in large fermenters. Microorganisms are subjected to fluctuating environmental conditions as they pass through the zones in a stirred vessel or along a closed loop fermenter.A 7-litre fermenter was modified to simulate the dissolved gas and hydrostatic pressure gradients in large vessels.The effect of cycling dissolved oxygen tension (DOT) on penicillin production by Penicillium chrysogenum P1 was investigated. The fermentation was affected by evironmental conditions such as medium composition, pH, size of inoculum, stirrer speed and DOT. Inoculum size below 10% (v/v) and stirrer speeds above 850 rpm caused significant reductions in specific prenicillin production rates (q_pen). q_pen values were measured at different constant DOT levels. Below 30% air saturation q_pen decreased sharply and no production was observed at 10%. Penicillin synthesis was impaired irreversibly below 10% DOT. The same profile was observed at higher stirrer speeds and air flow rates indicating that the effect was a physiological one. Oxygen uptake of the culture was affected significantly below 7% DOT, demonstrating that the critical DOT values for penicillin production and oxygen uptake are two distinct parameters. Carrying out the fermentation at one atmosphere over pressure was found to have no effect. When the dissolved oxygen concentration of the culture medium was cycled around the critical DOT for penicillin production, a considerable decrease in the specific penicillin production rate was observed. The effect was reversible but not transient, indicating a shift in cell metabolism.These results demonstrate the unfavourable effect of fluctuating environmental conditions on culture performance in stirred tanks. They suggest that these effects should be accounted for during strain selection, process development and scale up stages of an industrial process if the productivities in small scale vessels are to be obtained.     

Bubble coalescence behaviour in biological media

Summary A twin bubble column was used to measure the k_La values for oxygen in model and cultivation media using the steady state method described previously (Adler et al. 1980). Desmophen and soy oil were used as antifoam agents together with model and/or cultivation media for Chaetomium cellulotyticum, Trichoderma reesei, Hansenula polymorpha, Saccharomyces cerevisiae and Escherichia coli. The bubble coalescence behavior is mainly influenced by antifoam agents and somewhat by protein and alcohol additives. In the range investigated (0.01 to 0.1%.), the k_La values are not influenced by the Desmophen concentration and only slighthly by the soy oil concentration (0.5 to 1.5%.). The coalescence behaviour was characterized by the ratio m_corr=(k_La)_corr/(k_La)_ref. A nutrient salt solution with Desmophen was used as a reference. The k_La measured in the investigated media were corrected by considering the differences in k_La's in the investigated and reference media. These m_corr values can directly be used for bubble columns close to the optimum aeration rate.Symbols a Specific gas/liquid interfacial area - c Concentration - k_L Mass transfer coefficient - k_La Volumetric mass transfer coefficient - W_SG Superficial gas velocity - E_G Relative gas hold-up     

Enhancement of oxygen mass transfer in stirred bioreactors using oxygen-vectors. 1. Simulated fermentation broths

Oxygen mass transfer represents the most important parameter involved in the design and operation of mixing-sparging equipment for bioreactors. It can be described and analyzed by means of the mass transfer coefficient, k_La. The k_La values are affected by many factors such as geometrical and operational characteristics of the vessels, media composition, type, concentration and microorganism morphology, and biocatalysts properties. The efficiency of oxygen transfer could be enhanced by adding oxygen-vectors in broths, such as hydrocarbons or fluorocarbons, without increasing the energy consumption for mixing or aeration. The experimental results obtained for simulated broths indicated a considerable increase of k_La in the presence of n-dodecane, and the existence of a certain value of n-dodecane concentration that corresponds to a maximum mass transfer rate of oxygen. The magnitude of the positive effect of n-dodecane depends both on the broths characteristics and operational conditions of the bioreactor.Notation d stirrer diameter, mm - d oxygen electrode diameter, mm - D bioreactor diameter, mm - h distance from the inferior stirrer to the bioreactor bottom, mm - H bioreactor height, mm - k_La oxygen mass transfer coefficient, s^-1 - l impeller blade length, mm - I oxygen electrode immersed length, mm - P power consumption for mixing of non-aerated broths, W - P_a power consumption for mixing of aerated broths, W - (P_a/V) specific power input, W/m³ - s baffle width, mm - v_S superficial air velocity, m/s - V volume of medium, m³ - w impeller blade height, mm - volumetric fraction of oxygen-vector - _a apparent viscosity, Pa*s - density, kg/m³     

Mixing and oxygen transfer characteristics of a microplate bioreactor with surface-attached microposts

Bioprocess optimization for cell-based therapies is a resource heavy activity. To reduce the associated cost and time, process development may be carried out in small volume systems, with the caveat that such systems be predictive for process scale-up. The transport of oxygen from the gas phase into the culture medium, characterized using the volumetric mass transfer coefficient, k_La, has been identified as a critical parameter for predictive process scale-up. Here, we describe the development of a 96-well microplate with integrated Redbud Posts to provide mixing and enhanced k_La. Mixing in the microplate is characterized by observation of dyes and analyzed using the relative mixing index (RMI). The k_La is measured via dynamic gassing out method. Actuating Redbud Posts are shown to increase rate of planar homogeneity (2 min) verse diffusion alone (120 min) and increase oxygenation, with increasing stirrer speed (3500-9000 rpm) and decreasing fill volume (150-350 μL) leading to an increase in k_La (4-88 h⁻¹). Significant increase in Chinese Hamster Ovary growth in Redbud Labs vessel (580,000 cells mL^-1) versus the control (420,000 cells mL^-1); t(12.814) = 8.3678, p ≤ .001), and CD4⁺ Naïve cell growth in the microbioreactor indicates the potential for this technology in early stage bioprocess development and optimization.     

Mathematical model based estimation of volumetric oxygen transfer coefficient(s) in the production of proteolytic enzymes in Bacillus amyloliquefaciens

Alkaline protease is a class of important hydrolytic enzymes having wide applications in bioprocess industries. Their optimum pH in the alkaline range and stability at higher temperatures make them ideal in detergent and leather processing industries. These enzymes have excellent depilating capacity. The present study aims at process optimization for the production of alkaline protease from Bacillus amyloliquefaciens ATCC 23844. Information on the optimal operating temperature and pH were elicited from specific growth rates and alkaline protease yields. It was also observed that besides pH and temperature, the oxygen transfer rate is another important limiting variable for the production of protease. Volumetric oxygen transfer coefficient (k _L a) was estimated at various impeller speeds and aeration rates. The optimal impeller speed and aeration rates were determined from k _L a and the relative protease yield data. It was understood that the oxygen transfer rate is one of the crucial parameters for the production of proteolytic enzymes by B. amyloliquefaciens.     

Dissolved oxygen as principal parameter for conidia production of biocontrol fungi Trichoderma viride in non-Newtonian wastewater

Dissolved oxygen (DO) concentration was selected as a principal parameter for translating results of shake flask fermentation of Trichoderma viride (biocontrol fungi) to a fermenter scale. All fermentations were carried out in a 7.5 l automated fermenter with a working volume of 4 l. Fermentation performance parameters such as volumetric oxygen transfer coefficient (k _L a), oxygen uptake rate (OUR), rheology, conidia concentration, glucose consumption, soluble chemical oxygen demand, entomotoxicity and inhibition index were measured. The conidia concentration, entomotoxicity and inhibition index were either stable or improved at lower DO concentration (30%). Variation of OUR aided in assessing the oxygen supply capacity of the fermenter and biomass growth. Meanwhile, rheological profiles demonstrated the variability of wastewater during fermentation due to mycelial growth and conidiation. In order to estimate power consumption, the agitation and the aeration requirements were quantified in terms of area under the curves, agitation vs. time (rpm h), and aeration vs. time (lpm h). This simple and novel strategy of fermenter operation proved to be highly successful which can be adopted to other biocontrol fungi.     

Cellulose degradation and carboxymethylcellulase production by Sporocytophaga myxococcoides grown in an air-lift fermenter

Summary Sporocytophaga myxcoccoides was grown in a 31 air-lift fermenter using a medium containing 2% w/v insoluble cellulose. The cellulose content of the medium reduced the k_La of the fermenter but during growth the dissolved oxygen concentration did not fall below 75% saturation. Rates of cellulose degradation and extracellular enzyme production were similar to those reported for a stirred-tank fermenter.     

Determination of the overall volumetric mass transfer coefficient kLa,by a temperature dependent change of gas solubility

Summary The solubility of oxygen in the liquid phase of a bioreactor was changed by a ramp change of temperature, and k_La was determined from the resulting return to equilibrium of dissolved oxygen activity. The maximum k_La that can be measured by this method in a standard laboratory scale bioreactor is 145 h^–1 corresponding to a temperature change rate of 320°C h^–1.Nomenclature p Difference between p_G and p_L (% saturation) - T Ramp change of temperature (°C) - E Temperature-compensated output from the oxygen electrode (A) - E_u Uncompensated output from the oxygen electrode (A) - k_La Overall volumetric mass transfer coefficient (h^–1) - k_La_Tm Overall volumetric mass transfer coefficient at temperature T_m (h^–1) - P_G Dissolved oxygen activity in equilibrium with the gas phase (% saturation) - p_L Dissolved oxygen activity (% saturation) - p_Lm Dissolved oxygen activity at time t_m (% saturation) - t Time (h) - t_m Time of maximum p (h) - T Temperature (°C) - T_cal Calibration temperature of the oxygen electrode (°C) - T_m Final temperature after a temperature shift (°C) - T_n Temperature at time t_n     

Mass cultivation of Medicago sativa growing on lactose: Kinetic aspects

Summary Batch cultures of Medicago sativa cells have been carried out in the dark under aerobic conditions using lactose as the sole carbon source. The stoichiometric analysis has been correlated with both the oxygen demand and the cell productivity in an oxygen-limited cultivation. The minimum oxygen transfer has been estimated to be 12.5 h^–1, i.e., 0.3 v.v.m; this initial aeration rate led to cell necrosis. Starting with a low oxygen transfer coefficient k_L·a and increasing the air flow rate during the course of fermentation gave an exponential growth phase. The maximum specific growth rate was 0.012 h^–1 and the growth yield was 0.43 g.d.w./g. of lactose. On the basis of the mass-balance relation the maintenance coefficient and the maximum growth yield have been calculated.     

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.     

Sulphite oxidation in the disc fermenter

Summary Sulphite oxidation in the disc fermenter showed an initial peak in the oxygen absorption rate followed eventually by a much lower constant final rate. The reaction was also found not to be diffusion controlled and therefore not suitable for measurement of true physical values of k_La.     

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     

Bubble coalescence behaviour in biological media

Summary Volumetric mass transfer coefficients (k_La) were measured by a steady state method in a twin bubble column to characterize the coalescence behaviour of the medium. Employing Hansenula polymorpha cultivation broths, k_La values were compared with those measured in model media in the presence and absence of antifoam agents. The ratio of the volumetric mass transfer coefficient in the system investigated to that in water, , was employed to characterize the cultivation medium.Symbols a Specific gas/liquid interfacial area with regard to the liquid volume in reactor - d_e Dynamical equilibrium bubble diameter - d_H Perforated plate hole diameter - d_p Primary bubble diameter - d_S Sauter bubble diameter - F_v Liquid feed rate - H Bubbling layer height - k_L Gas/liquid mass transfer coefficient - k_La Volumetric mass transfer coefficient - m k_La/(k_La)_r coalescence index - m_corr Corrected coalescence index [Eq. (3)] - OTR Oxygen transfer rate - P_O Dissolved O₂-partial pressure in BS2 - P₁ Dissolved O₂-partial pressure in BS1 - P_O P_O/P_S relative oxygen saturation in BS2 - P₁ P₁/P_S relative oxygen saturation in BS1 - P_S Saturation dissolved oxygen partial pressure - R_c dn_B/dt coalescence rate - S Substrate concentration - t_F Time since the beginning of the cultivation - X Biomass concentration - V₁ Liquid volume in BS1 - w_SG Superficial gas velocity in BS1 - G Gas holdup in BS1 - ₁ V₁/F_v mean liquid residence time in BS1 - BS1 O₂ absorber column - BS2 O₂ desorber column - D Desmophen (antifoam agent) - NS Nutrient salt solution (Table 1)     

Optimization of lipase production by <Emphasis Type="Italic">Staphylococcus warneri</Emphasis> EX17 using the polydimethylsiloxanes artificial oxygen carriers

In this research, the combined effects of polydimethylsiloxane (PDMS) and different conditions of oxygen volumetric mass transfer coefficient (k_La) on lipase production by Staphylococcus warneri EX17 were studied and optimized in bioreactor cultures. Raw glycerol from biodiesel synthesis was used as the sole carbon source. Full-factorial central composite design and the response surface methodology were employed for the experimental design and analysis of the results. The optimal polydimethylsiloxane concentration and mass coefficient transfer (k_La) were found to be 13.5% (v/v) and 181 h⁻¹, respectively. Under these conditions, the maximal cell production obtained was 10.0 g/l, and the volumetric lipase activities of approximately 490 U/l, after 6 h of cultivation. These results are in close agreement with the model predictions. Results obtained in this work reveal the positive effects of PDMS on oxygen volumetric mass transfer coefficient (k_La) in the Staphylococcus warneri EX17 cultivation and lipase production.