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.     

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.     

Modeling and simulation of oxygen-limited partial nitritation in a membrane-assisted bioreactor (MBR)

Combination of a partial nitritation process and an anaerobic ammonium oxidation process for the treatment of sludge reject water has some general cost-efficient advantages compared to nitrification-denitrification. The integrated process features two-stage autotrophic conversion of ammonium via nitrite to dinitrogen gas with lower demand for oxygen and no external carbon requirement. A nitrifying membrane-assisted bioreactor (MBR) for the treatment of sludge reject water was operated under continuous aeration at low dissolved oxygen (DO) concentrations with the purpose of generating nitrite accumulation. Microfiltration was applied to allow a high sludge retention time (SRT), resulting in a stable partial nitritation process. During start-up of the MBR, oxygen-limited conditions were induced by increasing the ammonium loading rate and decreasing the oxygen transfer. At a loading rate of 0.9 kg N m(-3) d(-1) and an oxygen concentration below 0.1 mg DO L(-1), conversion to nitrite was close to 50% of the incoming ammonium, thereby yielding an optimal effluent within the stoichiometric requirements for subsequent anaerobic ammonium oxidation. A mathematical model for ammonium oxidation to nitrite and nitrite oxidation to nitrate was developed to describe the oxygen-limited partial nitritation process within the MBR. The model was calibrated with in situ determinations of kinetic parameters for microbial growth, reflecting the intrinsic characteristics of the ammonium oxidizing growth system at limited oxygen availability and high sludge age. The oxygen transfer coefficient (K(L)a) and the ammonium-loading rate were shown to be the appropriate operational variables to describe the experimental data accurately. The validated model was used for further steady state simulation under different operational conditions of hydraulic retention time (HRT), K(L)a, temperature and SRT, with the intention to support optimized process design. Simulation results indicated that stable nitrite production from sludge reject water was feasible with this process even at a relatively low temperature of 20 degrees C with HRT down to 0.25 days.     

Studies on Oxygen Transfer in Submerged Fermentations

This paper refers to the application of gas analyzers for the determination of oxygen transfer rate, showing examples in the studies and the performances of submerged fermentations. Oxygen and carbon dioxide analyzers were set to monitor the gas streams to and from the fermentor. Continuous data on the concentrations of oxygen and carbon dioxide in the air streams were thus provided throughout the fermentation. Distinctive characters of this method were applicability to fermentors in practice and ability of obtaining data directly relating to the fermentations.

The modification of sulfite oxidation method for the determination of oxygen transfer rate from air into liquid or of a measure of aeration effectiveness was made. The proposed method was the application of gas analyzers in the studies on submerged fermentation. Some comparative discussions were made between this and the conventional titrimetric method. This modified method could be applied to biological systems with no alteration, therefore, it was made possible to compare the sulfite solution with the biological systems in relation to the problems on oxygen transfer.     

Effects of cultivation conditions on the production of natamycin with Streptomyces gilvosporeus LK-196

Natamycin is a very attractive antifungal agent with wide applications in medical and food industries. In order to improve the productivity of natamycin, the effects of cultivation conditions were investigated with Streptomyces gilvosporeus LK-196 in the shake flasks and 30-L fermentors. The results showed that dissolved oxygen and shear force would affluence the biosynthesis of natamycin significantly. The high concentration of natamycin (2.03g/L) was achieved under the suitable culture conditions in the shake flask scale. Further investigations in 30-L fermentors showed that the optimal pH was controlled at 6.0 during the whole bioprocess, and the dissolved oxygen level should be more than 30% by adjusting the aeration and agitation rates for high production of natamycin. Under these optimal conditions the high concentration of natamycin (3.94g/L) was achieved with Str. gilvosporeus LK-196 in the 30-L fermentor. Finally, the high-level fermentation process was successfully scaled up to 1000-L fermentors and 18,000-L fermentors in the pilot plant.     

Approaches to KL a measurements in solid state fermentation

Summary A convenient method for measuring K_L a in a solid state medium is proposed. Due to the particular nature of the substrate used in solid state fermentation, different modifications of the sulfite oxidation method have been necessary. This first approach allows to study the influence of air inflow rate and dry matter percentage of the medium on the oxygen volumetric mass transfer coefficient.     

The Dissolved Oxygen and Respiration Rates in Pellicle and Submerged Cultures of Corynebacterium diphtheriae

S ummary . The concentration of dissolved oxygen in submerged and static cultures of Corynebacterium diphtheriae was measured with a cathode ray polarograph. In a pellicle culture of the type frequently used for production of diphtheria toxin the concentration of dissolved oxygen in the medium below the pellicle fell sharply after the third or fourth day, and growth then proceeded in a medium which contained only minimal amounts of dissolved oxygen, in spite of the fact that only a cotton wool plug separated the atmosphere above the medium from the outside air.
The measurement of dissolved oxygen in submerged cultures was applied to the determination of respiration rates in growing cultures. The respiration rate for the test organisms varied at different stages in the period of a submerged batch culture. The rate was greatest at the start of the logarithmic phase, and thereafter declined until a steady state was reached.     

Critical assessment of gassing-in methods for measuring k(l)a in fermentors

The reliability of dynamic measurement methods of k(l)a in fermentors using a step oxygen concentration change in the feed gas was tested. The tests were performed both for the original variant using the nitrogen right harpoon over left harpoon air exchange and the newly presented variant using the oxygen-enriched air (27 vol % O(2)) --> air exchange. The testing consisted in comparing k(l)a values determined from these methods with values determined from the steady-state Na(2)SO(3) feeding method and the dynamic pressure method, the reliability of which was proven earlier. The measurements were done in water (coalescent batch) and in 0.5M Na(2)SO(4) solution with and without the addition of 1 wt % carboxymethylcellulose (noncoalescent batches). It was found that in noncoalescent liquids the methods tested give extremely low k(l)a values (as low as 15% of the correct value). The methods are defective in principle irrespective of the gases used for exchange.     

Oxygen transfer kinetics of riboflavin fermentation by Ashbya gossypii in agitated fermentors

Effects of agitation and aeration rates on volumetric oxygen transfer coefficient and oxygen uptake rate of a riboflavin broth containing Ashbya gossypii were investigated in three batch, sparged, and agitated fermentors having the working volumes of 0.42, 0.85, and 2.5 l. The change of oxygen uptake rate with time at 250 rev min⁻¹ stirring and vvm aeration rates was shown. The volumetric oxygen transfer coefficients and maximum oxygen uptake rates obtained have been correlated to mechanical power inputs per unit volume of the fermentation broth and the superficial air velocities.     

Use of the glucose oxidase system to measure oxygen transfer rates

This investigation used the glucose oxidase system to simulate oxygen transfer rate in fermentation broths. It was demonstrated that the fungal preparation contained sufficient lactonase activity so that D -glucono-δ-lactone did not accumulate and that the rate of production of gluconic acid was proportional to the oxygen uptake rate. Enzyme concentrations of 1.5–2 g/1 were found adequate to determine oxygen absorption rates in shake flasks while maintaining the dissolved oxygen concentration of low levels. The apparent Michaelis constant for oxygen, K_m(O₂), was found to be 27% saturation with air; this value along with experimentally determined uptake rates could be used to calculate dissolved oxygen concentration in lieu of using a dissolved oxygen probe. Enzyme concentrations of 5 g/l were sufficient to give linear acid production and low dissolved oxygen concentrations in a bench-scale fermenter with no foaming or enzyme deactivation. The method is considered more valid and easier to employ than previously utilized techniques such as sulfite oxidation. Extension of the system to evaluating aeration effectiveness and scaleup of fermentation equipment is discussed.     

Rapid large-scale growth of Helicobacter pylori in flasks and fermentors.

We developed procedures for large-scale cultivation of Helicobacter pylori in flasks and fermentors. Flasks incubated closed under a microaerophilic gas phase with a cotton plug covered by a plastic bag, followed by removal of the bag after 8 h, gave excellent growth. Growth in a 10-liter fermentor led to excessive foaming if the medium was sparged with gas; silicone- or polyglycol-based antifoaming agents were severely inhibitory. Use of fermentor surface gassing, first with a microaerophilic 6% oxygen gas mixture, then with air, and then with 95% oxygen, allowed the culture to grow to an A600 of 2.5 in < 24 h. This method was modified for scale-up to a 100-liter fermentor.     

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.     

以摇瓶所得摄氧率为基准进行发酵放大

通过设计特殊摇瓶,用亚硫酸盐法测出摇瓶口纱布层氧通透率的基础上,在实际发酵情况下通过测定瓶内气、液相氧的变化得出其发酵过程中的摄氧率(OUR)及氧传递系数(K_La)。以特制摇瓶取得的菌体CUR为基准进行发酵过程和发酵罐的放大。通过质谱仪在线检测及采样分析,研究了3种不同供气流量及搅拌转速下的放大结果。摇瓶与发酵罐在菌体OUR、菌体产量方面吻合很好,而在整个放大过程中,发现摇瓶与发酵罐内的氧传递系数(K_La)、溶解氧(C_L)差异较大。     

The effect of dissolved oxygen on the metabolic profile of a murine hybridoma grown in serum-free medium in continuous culture

The murine B-lymphocyte hybridoma, CC9C10 was grown at steady state under serum-free conditions in continuous culture at dissolved oxygen (DO) concentrations in the range of 10% to 150% of air saturation. Cells could be maintained with this range at high viability in a steady state at a dilution rate of 1 d(-1), although with lower cell concentrations at higher DO. A higher specific antibody production measured at higher DO was matched by a decrease in the viable cell concentration at steady state, so that the volumetric antibody titre was not changed significantly. An attempt to grow cells at 250% of air saturation was unsuccessful but the cells recovered to normal growth once the DO was decreased.There was a requirement for cellular adaptation at each step-wise increase in dissolved oxygen. Adaptation to a DO of 100% was associated with an increase in the specific activities of glutathione peroxidase (x18), glutathione S-transferase (x11) and superoxide dismutase (x6) which are all known antioxidant enzymes. At DO above 100%, the activities of GPX and GST decreased possibly as a result of inactivation by reactive oxygen radicals.The increase in dissolved oxygen concentration caused changes in energy metabolism. The specific rate of glucose uptake increased at higher dissolved oxygen concentrations with a higher proportion of glucose metabolized anaerobically. Short-term radioactive assays showed that the relative flux of glucose through glycolysis and the pentose phosphate pathway increased whereas the flux through the tricarboxylic acid cycle decreased at high DO. Although the specific glutamine utilization rate increased at higher DO, there was no evidence for a change in the pattern of metabolism. This indicates a possible blockage of glycolytic metabolites into the TCA cycle, and is compatible with a previous suggestion that pyruvate dehydrogenase is inhibited by high oxygen concentrations.Analysis of the oxygen uptake rate of cell suspensions at steady state under all conditions showed a pronounced Crabtree effect which was manifest by a decrease (up to 40%) in oxygen consumption on addition of glucose. This indicates that the degree of aerobic metabolism in these cultures is highly sensitive to the glucose concentration. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 153-164, 1997.     

Analysis of biological wastewater treatment processes using multicomponent gas phase mass balancing.

A reactor system using off-gas analysis was developed for analyzing wastewater treatment process reactions. Using a mass spectrometer for the gas analysis provides the ability to simultaneously measure several gas components (such as oxygen, nitrogen, carbon dioxide, and argon). One of the benefits of the reactor design was the precise control of the dissolved oxygen concentration, uncoupled from the system turbulence, which was controlled via a gas recycle loop. This feature allowed control of the turbulence within the reactor without any need for mechanical stirring. Using oxygen as the test gas, the reactor was shown to perform well in the measurement of oxygen uptake rate of nitrifying activated sludge. The oxygen uptake rate calculations were made using a simple calibration method developed for the reactor system. The reactor was able to provide precise and accurate results for this test case. Furthermore, the system was capable of measuring under dynamic process conditions, as well as when the process rates were constant (steady state).     

On-line continuous measurement of the oxygen consumption rate in mammalian cell culture

A method for on-line, continuous measurement of the oxygen consumption rate (Io₂) by mammalian cells on a microcarrier was developed and its reliability investigated. Utilizing the periodic dissolved oxygen (DO) fluctuation in the normal on-off control of DO, on-line, continuous measurement of Io₂ was carried out in which real-time estimation of the DO saturation concentration was made by measuring the gas-phase pressure and the gas-phase oxygen concentration. It was found that the continuously measured Io₂ value was quantitatively exact and could be applied commercially using the oxidation reaction of glucose by glucose oxidase.     

Gasometric measurement of sulfite oxidation

In the commonly used sulfite method the consumption of sulfite is determined by iodometry. Since however, the addition of organic substances may interfere with iodometry (e.g. due to chemical reactions with iodine) the gasometric measurement of sulfite oxidation has been developed for analysis of how different culture media may influence the oxygen transfer rate. The striking decrease of sulfite oxidation rate due to addition of culture media to the sulfite solution suggests that adsorption of orgnic components in the gas liquid interface may account for an additional diffusion barrier and thus for a decrease of the oxygen transfer coefficient which in addition gives an explanation for differences between values found by the sulfite method and by aerobic cultivations. Consequently identical values of oxygen transfer rate have been obtained for both systems whenever the sulfite system has been properly adjusted to the aerobic cultivation conditions. In so far, the gasometric sulfite method proved to be a unique tool for rapid determination of factors influencing oxygen transfer rate in fermentation processes which may give rise to a reappraisal as to the relevance of the sulfite method for oxygen transfer optimization.     

High cell density induces spontaneous bifurcations of dissolved oxygen controllers during CHO cell fermentations

High cell density cultures of CHO cells growing in a bioreactor under dissolved oxygen control were found to undergo spontaneous bifurcations and a subsequent loss of stability some time into the fermentation. This loss of stability was manifested by sustained and amplified oscillations in the bioreactor dissolved oxygen concentration and in the oxygen gas flow rate to the reactor. To identify potential biological and operational causes for the phenomenon, linear stability analysis was applied in a neighborhood of the experimentally observed bifurcation point. The analysis revealed that two steady state process gains, K(P1) and K(P2), regulated k(l)a and gas phase oxygen concentration inputs, respectively, and the magnitude of K(P1) was found to determine system stability about the bifurcation point. The magnitude of K(P1), and hence the corresponding open-loop steady state gain K(OL1), scaled linearly with the bioreactor cell density, increasing with increasing cell density. These results allowed the generation of a fermentation stability diagram, which partitioned K(C)-N operating space into stable and unstable regions separated by the loci of predicted critically stable controller constants, K(C,critical), as a function of bioreactor cell density. This consistency of this operating diagram with experimentally observed changes in system stability was demonstrated. We conclude that time-dependent increases in cell density are the cause of the observed instabilities and that cell density is the critical bifurcation parameter. The results of this study should be readily applicable to the design of a more robust controller.     

A study of mass transfer in yeast in a pulsed baffled bioreactor

We report experimental data of mass transfer of oxygen into yeast resuspension in a pulsed baffled bioreactor. The bioreactor consists of a 50-mm-diameter column with the presence of a series of either wall (orifice) or central (disc) baffles or a mixture of both where fluid oscillation can also be supermposed during the experiments. Air bubbles are sparged into the bottom of the pulsed baffled bioreactor, and the kinetics of liquid oxygen concentration in the yeast solution is followed using a dissolved oxygen probe with a fast response time of 3 s together with the dynamic gassing-out technique. Among the three different baffle geometries investigated, the orifice baffles gave the highest and sharpest increase in the oxygen transfer rate, and the trends in the k(L)a measurements are consistent with the fluid mechanics observed within both the systems and previous work. In addition, we have also compared the k(L)a values with those obtained in a stirred tank; an 11% increase in the K(L)a is reported. (c) 1995 John Wiley & Sons, Inc.     

Effect of Oxygen Tension, Mn(II) Concentration, and Temperature on the Microbially Catalyzed Mn(II) Oxidation Rate in a Marine Fjord

We present evidence that the oxidation of Mn(II) in a zone above the O₂/H₂S interface in the water column of Saanich Inlet, British Columbia, Canada, is microbially catalyzed. We measured the uptake of ⁵⁴Mn(II) in water samples under in situ conditions of pH and temperature and in the presence and absence of oxygen. Experiments in the absence of oxygen provided a measure of the exchange of the tracer between the dissolved and solid pools of Mn(II); we interpret the difference between experiments in the presence and absence of oxygen to be a measure of Mn(II) oxidation. Using this method we examined the effect of oxygen tension, Mn(II) concentration, and temperature on the initial in situ Mn(II) oxidation rate (V₀). Mn(II) oxidation was almost twice as fast under conditions of 67% air saturation (V₀=5.5 nM h⁻¹) as with the in situ concentration of 15 μM (5% air saturation; V₀=3.1 nM h⁻¹). Additions of ca. 18 μM Mn(II) completely inhibited all Mn(II) oxidation at three different depths in the oxidizing zone, and there was a temperature optimum for Mn(II) oxidation of around 20°C. These results are consistent with biologically mediated Mn(II) oxidation and indicate that the rate is limited by both oxygen and the concentration of microbial binding sites in this environment.