Optical method for the determination of the oxygen-transfer capacity of small bioreactors based on sulfite oxidation.

The growth of microorganisms may be limited by operating conditions which provide an inadequate supply of oxygen. To determine the oxygen-transfer capacities of small-scale bioreactors such as shaking flasks, test tubes, and microtiter plates, a noninvasive easy-to-use optical method based on sulfite oxidation has been developed. The model system of sodium sulfite was first optimized in shaking-flask experiments for this special application. The reaction conditions (pH, buffer, and catalyst concentration) were adjusted to obtain a constant oxygen transfer rate for the whole period of the sulfite oxidation reaction. The sharp decrease of the pH at the end of the oxidation, which is typical for this reaction, is visualized by adding a pH dye and used to measure the length of the reaction period. The oxygen-transfer capacity can then be calculated by the oxygen consumed during the complete stoichiometric transformation of sodium sulfite and the visually determined reaction time. The suitability of this optical measuring method for the determination of oxygen-transfer capacities in small-scale bioreactors was confirmed with an independent physical method applying an oxygen electrode. The correlation factor for the maximum oxygen-transfer capacity between the chemical model system and a culture of Pseudomonas putida CA-3 was determined in shaking flasks. The newly developed optical measuring method was finally used for the determination of oxygen-transfer capacities of different types of transparent small-scale bioreactors.     

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.     

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.     

Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation

Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, k(L)a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k(L)a values in one commercial reader were about 10 to 40 h(-1). k(L)a values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k(L)a values in a 96-well microtiter plate. The resulting values were about 130 h(-1), which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h(-1). Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions.     

Effects of the oxygen transfer rate on ferrous iron oxidation by Thiobacillus ferrooxidans

Ferrous iron oxidation by Thiobacillus ferrooxidans was studied in shake flasks and a bubble column under different aeration conditions. The maximum biooxidation rate constant was affected by oxygen transfer only at low aeration intensities. At oxygen transfer rates higher than 0.03 mmol O₂ l⁻¹ min⁻¹, the maximum biooxidation rate constant was about 0.050 h⁻¹ in both shake flasks of different size and the bubble column. The oxygen transfer rate could be used as a basis for scaling up bioreactors for ferrous iron biooxidation by T. ferrooxidans.     

Combined sulfite method for the measurement of the oxygen transfer coefficient k(L)a in bio-reactors

The combined sulfite method is proposed for the measurement of oxygen transfer coefficients, k_La, in bioreactors. The method consists of a steady-state and a dynamic measurement which are carried out under the same experimental conditions and thus yield data for both methods during one experiment. The applied experimental conditions are shown to avoid chemical enhancement during the steady-state measurement. Moreover, no parallel sulfite oxidation occurs during the oxygen saturation phase of the dynamic measurement. Under the applied experimental conditions, no information about the sulfite oxidation kinetics is required and possible metal ion impurities in sulfite salts do not influence the measurement. The characterization of a laboratory-scale bioreactor aerated with pure oxygen yields k_La values during the steady-state and the dynamic measurements that are in good agreement with the dynamic pressure method, the correctness of which is generally accepted. When air is used for absorption, the steady-state measurement yields k_La values that correlate to the correct variant of the standard dynamic method. The dynamic measurement with air absorption yields a k_La value which considers the influence of the non-uniform bubble size distribution present in bubble-aerated bioreactors.     

On-line oxygen uptake rate and culture viability measurement of animal cell culture using microplates with integrated oxygen sensors

O2 uptake rates of animal cells (Chinese hamster ovary-CHO) were measured in 96-well microtiter plates by integrating with fluorescent sensors thereby measuring fluorescence intensity ratios of an O2-sensitive and an insensitive fluorophor. O2 consumption rate was estimated from measured dissolved O2 and from O2 mass transfer coefficient determined in advance. Specific uptake decreased with time from 3.2 x 10(-13) mol O2 cell(-1) h(-1) at 15 h cultivation to 1.8 x 10(-13) mol O2 cell(-1) h(-1) at 48 h. Specific O2 uptake was also determined by sampling from a spinner-flask culture giving identical values. A cell viability assay for cultures based on O2 measurements is described in which cells are incubated outside the fluorescence reader and then the dissolved O2 is measured only once at a fixed time after the start of incubation. This protocol can be directly applied for high-throughput measurements.     

The oxygen transfer rate influences the molecular mass of the alginate produced by Azotobacter vinelandii

The influence of oxygen transfer rate (OTR) on the molecular mass of alginate was studied. In batch cultures without dissolved oxygen tension (DOT) control and at different agitation rates, the DOT was nearly zero and the OTR was constant during biomass growth, hence the cultures were oxygen-limited. The OTR reached different maximum levels (OTR_max) and enabled to establish various relative respiration rates. Overall, the findings showed that OTR influences alginate molecular mass. The mean molecular mass (MMM) of the alginate increased as OTR_max decreased. The molecular mass obtained at 3.0 mmol l⁻¹ h⁻¹ was 7.0 times higher (1,560 kDa) than at 9.0 mmol l⁻¹ h⁻¹ (220 kDa). An increase in molecular mass can be a bacterial response to adverse nutritional conditions such as oxygen limitation.     

Online monitoring of oxygen in spinner flasks

We show the application of a novel optical on-line sensor fixed in spinner flasks for the online monitoring of dissolved O₂ concentrations during mammalian cell growth. Using this sensor that requires only minute changes to the flask to be made, we could determine the volumetric O₂ transfer coefficient as well as O₂ consumption rates. Under normal growth conditions the cells did not undergo O₂ limitation. Also, the transfer of O₂ from the atmosphere to the spinner flasks is influenced by the use of screw caps. The on-line measurement was further applied to determine the O₂ uptake rates which can then be used to monitor the metabolic state of the cells and also for online process monitoring.     

Enhanced in situ dynamic method for measuring KLa in fermentation media

The overall oxygen mass transfer coefficient (K_La) is often used as scale-up factor of fermentation systems. In fermenter scale-up, it is desired to achieve the same K_La values at the larger scale than the one that was obtained at a smaller scale during the development stage. It is therefore important to be able to measure K_La in situ during fermentation and to also determine the action to be taken to maintain its value at its design set point. These objectives can be obtained by measuring K_La using the dynamic method and enhancing the K_La information by immediately conducting a series of changes in agitation speed and/or aeration rate to determine the influence of these variables on K_La. This enhanced dynamic method is demonstrated with two filamentous microorganisms: Trichoderma reesei for the production of cellulase and Aspergillus niger for the production of citric acid. Two different types of bioreactor were used: a reciprocating plate bioreactor and a stirred (Rushton) bioreactor. It is shown that the proposed method can provide a simple way to measure the local variation of K_La and to adjust its value to its set point during the course of fermentation.     

Evaluation of oxygen transfer rates in stirred-tank bioreactors for clinical manufacturing

Several methods are available for determining the volumetric oxygen transfer coefficient in bioreactors, though their application in industrial bioprocess has been limited. To be practically useful, mass transfer measurements made in nonfermenting systems must be consistent with observed microbial respiration rates. This report details a procedure for quantifying the relationship between agitation frequency and oxygen transfer rate that was applied in stirred-tank bioreactors used for clinical biologics manufacturing. The intrinsic delay in dissolved oxygen (DO) measurement was evaluated by shifting the bioreactor pressure and fitting a first-order mathematical model to the DO response. The dynamic method was coupled with the DO lag results to determine the oxygen transfer rate in Water for Injection (WFI) and a complete culture medium. A range of agitation frequencies was investigated at a fixed air sparge flow rate, replicating operating conditions used in Pichia pastoris fermentation. Oxygen transfer rates determined by this method were in excellent agreement with off-gas calculations from cultivation of the organism (P = 0.1). Fermentation of Escherichia coli at different operating parameters also produced respiration rates that agreed with the corresponding dynamic method results in WFI (P = 0.02). The consistency of the dynamic method results with the off-gas data suggests that compensation for the delay in DO measurement can be combined with dynamic gassing to provide a practical, viable model of bioreactor oxygen transfer under conditions of microbial fermentation.     

Oxygen transfer phenomena in 48-well microtiter plates: determination by optical monitoring of sulfite oxidation and verification by real-time measurement during microbial growth

Oxygen limitation is one of the most frequent problems associated with the application of shaking bioreactors. The gas-liquid oxygen transfer properties of shaken 48-well microtiter plates (MTPs) were analyzed at different filling volumes, shaking diameters, and shaking frequencies. On the one hand, an optical method based on sulfite oxidation was used as a chemical model system to determine the maximum oxygen transfer capacity (OTR(max)). On the other hand, the Respiration Activity Monitoring System (RAMOS) was applied for online measurement of the oxygen transfer rate (OTR) during growth of the methylotropic yeast Hansenula polymorpha. A proportionality constant between the OTR(max) of the biological system and the OTR(max) of the chemical system were indicated from these data, offering the possibility to transform the whole set of chemical data to biologically relevant conditions. The results exposed "out of phase" shaking conditions at a shaking diameter of 1 mm, which were confirmed by theoretical consideration with the phase number (Ph). At larger shaking diameters (2-50 mm) the oxygen transfer rate in MTPs shaken at high frequencies reached values of up to 0.28 mol/L/h, corresponding to a volumetric mass transfer coefficient (k(L)a) of 1,600 1/h. The specific mass transfer area (a) increases exponentially with the shaking frequency up to values of 2,400 1/m. On the contrary, the mass transfer coefficient (k(L)) is constant at a level of about 0.15 m/h over a wide range of shaking frequencies and shaking diameters. However, at high shaking frequencies, when the complete liquid volume forms a thin film on the cylindric wall of the well, the mass transfer coefficient (k(L)) increases linearly to values of up to 0.76 m/h. Essentially, the present investigation demonstrates that the 48-well plate outperforms the 96-well MTP and shake flasks at widely used operating conditions with respect to oxygen supply. The 48-well plates emerge, therefore, as an excellent alternative for microbial cultivation and expression studies combining the advantages of both the high-throughput 96-well MTP and the classical shaken Erlenmeyer flask.     

Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture

The effect of oxygen transfer rate (OTR) on β-carotene production by Blakelsea trispora in shake flask culture was investigated. The results indicated that the concentration of β-carotene (704.1 mg/l) was the highest in culture grown at maximum OTR of 20.5 mmol/(l h). In this case, the percentage of zygospores was over 50.0% of the biomass dry weight. On the other hand, OTR level higher than 20.5 mmol/(l h) was found to be detrimental to cell growth and pigment formation. To elucidate the effect of oxidative stress on β-carotene synthesis, the accumulation of hydrogen peroxide during fermentation under different OTRs was determined. A linear response of β-carotene synthesis to the level of H₂O₂ was observed, indicating that β-carotene synthesis is stimulated by H₂O₂. However, there was an optimal concentration of H₂O₂ (2400 μM) in enhancing β-carotene synthesis. At a higher concentration of H₂O₂, β-carotene decreased significantly due to its toxicity.     

Effects of air pressure oscillation amplitude on oxygen transfer rate and biomass productivity in a solid-state fermenter

The modified sulfite oxidation method was adapted for estimation of the overall oxygen transfer rate in a pressure oscillating, solid-state fermenter. At 4.5 atm and 30 °C, the oxygen transfer rate reached 717 mmol kg^–1 initial dry matter h^–1 in this system against 37 mmol kg^–1 initial dry matter h^–1 in a static tray fermenter. At 30 °C and 3 atm, Azotobacter vinelandii grew on wheat straw and reached 4.7×10¹⁰ c.f.u. g^–1 substrate dry matter after 36 h, while only 8.2×10⁹ c.f.u. g^–1 substrate dry matter was obtained in a static tray system.     

Use of the glucose oxidase system for estimation of oxygen transfer rate in a solid-state bioreactor

The glucose oxidase system was adapted for estimation of the overall oxygen transfer rate in a periodic pressure oscillating, solid-state bioreactor. Enzyme concentration of 40 ml enzyme preparation L⁻¹ was found adequate to give linear gluconic acid production and attain maximal oxygen absorption rates. At 4 atm and 30°C, the oxygen transfer rate reached 892 mmol kg⁻¹ initial dry matter h⁻¹ in this system, while only 121 mmol kg⁻¹ initial dry matter h⁻¹ was obtained in a conventional static tray bioreactor.     

A simplified method for the cultivation of extreme anaerobic Archaea based on the use of sodium sulfite as reducing agent

The extreme sensitivity of many Archaea to oxygen is a major obstacle for their cultivation in the laboratory and the development of archaeal genetic exchange systems. The technique of Balch and Wolfe (1976) is suitable for the cultivation of anaerobic Archaea but involves time-consuming procedures such as the use of air locks and glove boxes. We describe here a procedure for the cultivation of anaerobic Archaea that is more convenient and faster and allows the preparation of liquid media without the use of an anaerobic chamber. When the reducing agent sodium sulfide (Na₂S) was replaced by sodium sulfite (Na₂SO₃), anaerobic media could be prepared without protection from oxygen outside an anaerobic chamber. Exchange of the headspace of serum bottles by appropriate gases was sufficient to maintain anaerobic conditions in the culture media. Organisms that were unable to utilize sulfite as a source for cellular sulfur were supplemented with hydrogen sulfide. H₂S was simply added to the headspace of serum bottles by a syringe. The use of H₂S as a source for sulfur minimized the precipitation of cations by sulfide. Representatives of 12 genera of anaerobic Archaea studied here were able to grow in media prepared by this procedure. For the extremely oxygen-sensitive organism Methanococcus thermolithotrophicus, we show that plates could be prepared outside an anaerobic chamber when sulfite was used as reducing agent. The application of this method may faciliate the cultivation and handling of extreme anaerobic Archaea considerably. Received: January 4, 2000 / Accepted: April 5, 2000     

Adaptation of the "Dynamic Method" for measuring the specific respiration rate in oxygen transfer systems through diffusion membrane

Monitoring the specific respiration rate (Q(O2)) is a valuable tool to evaluate cell growth and physiology. However, for low Q(O2) values the accuracy may depend on the measurement methodology, as it is the case in animal cell culture. The widely used "Dynamic Method" imposes serious difficulties concerning oxygen transfer cancellation, especially through membrane oxygenation. This paper presents an improved procedure to this method, through an automated control of the gas inlet composition that can minimize the residual oxygen transfer driving force during the Q(O2) measurement phase. The improved technique was applied to animal cell cultivation, particularly three recombinant S2 (Drosophila melanogaster) insect cell lines grown in a membrane aeration bioreactor. The average measurements of the proposed method reached 98% of stationary liquid phase balance method, taken as a reference, compared to 21% when the traditional method was used. Furthermore, this methodology does not require knowledge of the volumetric transfer coefficient k(L)a, which may vary during growth.     

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.