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Bioprocess Monitoring of Dissolved Oxygen Using a Computerized Pulsing Membrane Electrode Jyh-chern Chen and Henry Y. Wang* Department of Chemical Engineering, The University of Michigan, Ann Arbor, Michigan 48109
A membrane oxygen electrode usually suffers from long-term signal deterioration due to environmental factors such as changes in hydrodynamic conditions and alteration of membrane oxygen diffusivity due to fouling. These problems can theoretically be overcome by the use of the same oxygen electrode in a pulsing mode. The effects of stirring rate, viscosity of the culture media, and addition of antifoam agents on the direct reading of this pulsing oxygen electrode were investigated, and the results were compared to the traditional pseudo-steady-state operation of the electrode. With a pulsing period controlled a t 1 s and a rest period of longer than 5 min, the transient signal obtained can be quite stable, and it showed minimal interference from various environmental changes. Dissolved oxygen tension of a cephalosporin C fermentation was monitored using both this pulsing and the conventional pseudo-steady-state methods, and their readings were compared to those measured from an off-line newly calibrated oxygen electrode. The reading from the pulsed electrode showed more favorable agreement with that of the off-line measurement. The error encountered in using the conventional method of dissolved oxygen measurement could vary as much as 40 % in comparison with the actual dissolved oxygen tension during a fermentation process.
Introduction Dissolved oxygen measurement in bioprocesses was greatly simplified with the introduction of membranecovered oxygen electrodes in the mid-1950s (Clark et al., 1953). Various modifications of this oxygen electrode design have been developed and used frequently both in research and in industry. Essentially all of these membrane electrodesare operated on the same principle, which consists of oxygen diffusion across a polymeric membrane and a thin electrolytelayer to a noble-metal-basedcathode surface; here the oxygen is reduced if the cathode is at a sufficiently negative potential with respect to the anode. The potential can either be supplied externally as in the case of a polarographic electrode or achieved by establishing an internal fuel cell as in the case of a galvanic electrode. Current output of the electrode is directly proportional to the activity or partial pressure of the dissolved oxygen (Fatt, 1976). Theories and working principles of the oxygen electrode and its measurement have been extensively discussed and reviewed by various investigators such as Hitchman (1978) and Lee and Tsao (1979). Amperometric oxygen electrodesfor direct measurement of dissolved oxygen in liquids suffer from several disadvantages that include electrode output being dependent on the hydrodynamicconditionsof the liquid media (Linek, 1977) and varying with the changes of oxygen diffusivity through the membrane due to contamination or fouling (Buehler and Bucher, 1990). The sensitivity of the electrode also changes due to variations in membrane tension and membrane erosion. The flow dependence of the oxygen electrode can be minimized by using a thicker membrane or a cathode with smaller surface area. However, this will increase the response time and decrease the sensitivity of the electrode. It has been reported that the flow dependence of an oxygen electrode with a silicon/
* Author to whom correspondence should be addressed. 87567938/93/3009-0075$04.00/0
Teflon double membrane was 2-4 % ,but the response time was increased by 2-fold in comparison with that of a single Teflon membrane (Haddad, 1973). Chronoamperometry is an alternative approach to overcome some of the problems that limit the performance of an oxygen electrode (Lilley et al., 1969). This is achieved by applying a defined polarization potential during brief pulses and sampling the resulting transient current. At any interval of these pulses, the electrode is either opencircuited or at zero potential to stop the reduction of oxygen. This rest period lasts long enough for oxygen to diffuse and replenish the electrolyte layer and the membrane, allowing a thermodynamic equilibrium of oxygen to be reestablished at the cathode surface with the bulk solution. There are two types of charges in this transient current, namely, the capacitance charge and the Faradaic charge. The Faradaic part of the transient current arose from the rapid reduction of oxygen at the cathode surface during the pulsing period. Various methods of sampling and interpreting the transient current have been proposed (Mancy et al., 1962; Mancy, 1975; Langdon, 1984; Short and Shell, 1985;Kuessner, 1987; Bessetteet al., 1988; Wang and Li, 1989). The advantages of these pulsing methods of oxygen measurement include less interferencefrom flow conditions and membrane properties, higher oxygen sensitivity (Lilley et al., 19691, and lower temperature dependence (Wilson, 1974). Although many efforts have been described to improve the performance of these pulsing amperometric oxygen electrodes, there is little evidence on its practical application in bioprocess monitoring (Wang and Li, 1989). Dissolved oxygen is an important variable in various oxidative biological processes. The growth and metabolism of various microbial cultures usually alter with the dissolved oxygen concentration. In many traditional aerobic cultures, the oxygen electrode was often used to check whether the dissolved oxygen was controlled at a predetermined value. The accuracy of the measurement
0 1993 American Chemical Society and American Institute of Chemlcal Englneers
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was not of critical importance. On the other hand, many new bioprocesses require more stringent oxygen control and require the oxygen be controlled within 10% of a set value of low but defined oxygen tension (Buehler and Bucher, 1990). Therefore, there is still a need for longterm stable and accurate oxygen measuring devices in bioprocessing. Direct determination of dissolved oxygen in the fermentor using a pulsing amperometric electrode is evaluated in this article. The effects of stirring speeds, viscosity changes of the culture media, and the addition of an antifoam agent on the direct reading of this pulsing oxygen electrode were investigated. Foaming is a common phenomenon in many industrial fermentation processes. Addition of chemical antifoam agents may also cause severe fouling at the membrane surface of the electrode, thereby affecting the oxygen measurement.
Materials and Methods Reagents. All reagents used are commerciallyavailable reagent grade materials. The gold wire (10965,99.99 % ) and silver wire (11433, 99.9%) for the oxygen electrode are from Johnson-Matthey/AESAR Group (Ward Hill, MA). The silicone antifoam fluid (SAG 471) used is from Union Carbide Co. (St. Louis, MO). Oxygen Electrodes. Three polarographic oxygen electodes were constructed in our laboratory, and they were used throughout the experiment. The cathode was made by placing a segment of gold wire with a diameter of 1.0 mm and length of 5.0 mm within a glass capillary tube (3.0 mm 0.d.) after having been bonded to a silver wire. The glass around the unattached end was drawn over the gold segment, and the tip was polished so that only a disk of gold was available. A silver wire of diameter 0.5 mm was wound around the glass tube and used as an anode. The electrodes were covered by a Teflon membrane (Teflon PFA, fluorocarbon film lOOLP, E. I. du Pont de Nemours and Co., Inc., Wilmington, DE) with a thickness of 1.3 X cm and sheathed by an outer glass tube of diameter 6.0 mm. The space between the membrane inner surface and the face of the electrode was filled with a 2.0 N potassium chloride electrolyte solution. The oxygen electrode was protected by a polycarbonate tube when it was submerged in the fermentor. The oxygen electrode was sterilized by inserting the electrode in a 95% (v/v) ethanol solution for 24 h prior to the start-up of fermentation. Pulsing and Steady-StateTechniques. Steady-state and pulsing polarizing potentials (-0.7 V vs the silver anode) were applied, respectively, to the cathode of two oxygen electrodes by a potentiostat (chemicalmicrosensor, Diamond General Development Corp., Ann Arbor, MI). Pulsing potential was generated by switching on and off the circuit between the potentiostat and the electrodes via a terminal board (Model STA-01, Metra Byte Corp., Taunton, MA) and a relay board (Model ERA-01, Metra Byte). To avoid any reduction of oxygen by capacitive charge (Short and Shell, 1985), the potential in between the pulses was switched to -0.1 to 0.0 V. The resulting transient and steady-state currents were sampled by a multiplexor (Model EXP-16, Metra Byte) and converted to digital signals by an AD converter (DASH-8, Metra Byte). A portable personal computer (COMPAQ Corp., Houston, TX) and user friendly software were used to control the operation of measurement of dissolved oxygen. The software was developed to perform calibration, measurement, and database management for oxygen sensors using pulsing and steady-state techniques.
Microorganism, Media, and Culture Conditions. Cephalosporium acremonium strain CW 19, ATCC 36225 (American Type Culture Collection, Rockwell, MD), was maintained on agar slopes (Shen et al., 1986) and stored at 4 OC. The compositions of the seed medium and the fermentation medium are the same as the complex medium and defined chemical medium, respectively, described by Shen et al. (1986). Cells from a slant were suspended in 5 mL of sterile water and added to 40 mL of complex seed medium. Cultures were grown for 2-3 days in a rotary incubator/shaker at 25 "C and 250 rpm. Fermentations were started by inoculating 15 mL of seed culture into a l-L fermentor (BIOFLO, Model C30, New Brunswick Scientific Co., Inc., New Brunswick, NJ) containing 550 mL of defined fermentation medium (Shen et al., 1986). Agitation was provided by a motor which was magnetically coupled to a four-blade impeller inside the vessel, which had an adjustable speed from 50 to loo0 rpm. Humid air was supplied to the vessel through a flow meter and an air filter. Temperature was controlled at 25 f 0.2 "C (Smith, 1985) by a solid-state controller with a thermistor and a heater element inside the vessel. The pH of the fermentation medium was maintained at pH 7.0 f 0.1 (Smith, 1985) by a autoclavable pH electrode (Cole-Parmer Instrument Co., Chicago, IL) and a pH controller (Model 5997-20, Cole-Parmer), which controlled the addition of 2 N NaOH solution. Measurement of Dissolved Oxygen in the Fermentation Broth. The dissolved oxygen in the fermentation medium was monitored simultaneously by a pulsing amperometric oxygen electrode and a steady-state polarized oxygen electrode. A hooded sampler is attached to a sampling tube that extends to the bottom of vessel. The 50-mL screw-capped container of the sampler serves for intermittent collection and removal of culture samples. A newly calibrated steady-state polarized oxygen electrode was installed in the container for off-line dissolved oxygen analysis. Three 30-mL samples were taken separately during the fermentation. Calibrations of the oxygen electrode were made in distilled water with aeration of gas of known oxygen concentration. The dissolved oxygen concentration in the distilled water was measured by the modified Winkler's method (Thomas and Chamberlin, 1967).
Results and Discussion Comparison of Pulsing versus Steady-State Measurementof Dissolved Oxygen. Since the rate of oxygen reduction at the cathode surface is very rapid, the output current of the oxygen electrode is determined by the rate of oxygen diffusion through the membrane, the electrolyte, and other possible diffusion barriers such as fouling. In most agitated liquid systems, the electrode surface is covered by a thin layer of stagnant liquid, and this creates an additional diffusion barrier. Various mathematical models have been proposed to describe membrane-covered oxygen electrodes based on different geometries and operating conditions (Hitchman, 1978;Lee and Tsao, 1979; Myland and Oldham, 1984; Wang, and Li, 1989). Generally, the governing equation for the steady-state current of an oxygen electrode is given by:
i, = 4FAPo1/[(a/D$I,)+ (b/DmHm)+
-
( c / D P J+ (d/DBI,)l t a3 (1) This equation has the form of a current (is) flowing through several resistors in series arising from various diffusion barriers, and this current, is, is proportional to the oxygen
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1.61.64
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Dissolved oxygen concentration [ppml
Figure 1. Calibrationcurves of pulsing and steady-stateoxygen electrodes in distilled water at 25 "C.
tension (Poz)by a constant 4 multiplied by the Faraday constant (F)and cathode surface area (A). The resistances from the electrolyte (a/D$I,) and membrane ( b / D m H m ) should remain constant throughout the calibration and subsequent dissolved oxygen measurement. The resistances from fouling of the membrane (c/DfHf)and the stagnant liquid film thickness (d/D$I,), however, will change with any changes in the physicochemicalproperties of the liquid media and/or the flow velocity passing over the electrode surface. The steady-state current becomes a function of these diffusion barriers and their changes with time. The equation for the transient current is given by:
it = 4FAPozH&e'/2/r1/2t'f2
t
-
0
Provided with adequate sampling time, the transient current generally shows much higher oxygen sensitivity than the steady-state current (i.e., it > is). The transient current depends only on the properties of electrolyte film, which are more stable throughout the calibration, and the actual dissolved oxygen during the fermentation. Different combinations of pulsing periods from 0.5 to 5 s and rest periods from 0.5 to 10 min have been tested in the fermentor using our self-made polarographic oxygen electrode. There are various methods for sampling and interpreting the transient current (Mancy et al., 1962; Mancy, 1975; Langdon, 1984; Short and Shell, 1985; Kuessner, 1987;Bessette et al., 1988; Wang and Li, 1989). In this work, the transient current was taken as an average of 50 signals, sampling within 1 ms before the end of pulsing. With a pulsing period controlled at 1s and a rest period longer than 5 min, the transient current obtained was found to be reliable and free from any interference of the stirring rate. This time scheme was used throughout the following experiments. Calibrations of the oxygen electrodes using either the pulsing or steady-state method were initially performed in distilled water aerated with a gas stream of known oxygen concentration (Figure 1).Both calibration curves show fairly good linearity for dissolved oxygen concentration from 0 to 8.2 ppm (100% airsaturated distilled water at 1 atm and 25 "C). Figure 2 shows that the response of both electrodes changes synchronously with the changes of dissolved oxygen concentration in distilled water. The results indicate that both pulsing and steady-state oxygen electrodes show full agreement on the dissolved oxygen measurement in a wellstirred clean system.
0.2 0.1 0.01 0
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.
I
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.
.
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100
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150
.
I
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,
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Time [minute]
Figure 2. Responses of pulsing and steady-state oxygen electrodes upon the changes in dissolved oxygen concentration in distilled water at 25 "C.
Dependence of Oxygen Measurement on Hydrodynamic Conditions. The steady-state current output of an oxygen electrode depends on the thickness of the boundary layer; thereby current varied with any changes of the liquid viscosity and flow velocity of the fluid passing over the electrode. The effect of various stirring rates on the readings of a pulsing and a steady-state oxygen electrode in air-saturated distilled water is shown in Figure 3. The steady-state current changes cyclically between 0.22 and 0.46 pA when the stirring rate changes from 0 to 400 rpm. The transient current was not affected by these periodical changes. The current output of a steady-state oxygen electrode in air-saturated water increases with any increase in stirring rate and becomes stable only if the stirring rate exceeds 100 rpm (Figure 4). The effect of viscosity changes in the test solution on the performance of oxygen electrodes was examined using different amounts of dextran solutions (Figure 4). When both electrodes were put in air-saturated dextran solutions, the steady-state current decreased appreciably with any increase in dextran concentration. When the stirring rate was increased to 600 rpm, the oxygen electrode in 600 g/L dextran solution still exhibited a 35% decrease in ita steady-state current compared to that in distilled water. Similar behavior was observed by Linek (1977) using glycerol solution. The output current of a pulsing oxygen electrode only showed a slight decrease in the same viscous dextran solutions. Influence of Membrane Fouling on Oxygen Measurement. The buildup of fouling on the electrode membrane in the biological system usually arose from nutrient and cell debris adhesion to the membrane surface. The electrode current may drop to a very low value if live microbes adhere to the membrane surface. In most mycelial fermentations, fouling can easily occur due to rich media and long cultivation times. Foaming is another major problem encountered during many industrial fermentations. It is almost unavoidable since many media substitutes such as soya flour cause foaming when agitated or gassed. Foaming may also arise from the cell debris which causes late foam in many Actinomycete cultures. A most effective method to control foaming is to add a chemical antifoam agent such as a silicone oil based surfactant. Antifoam agents can adhere to the membrane surface and cause an increase of the overall diffusion resistance. Table I shows that the reading of a steady-state oxygen electrode in air-saturated distilled water decreased by 88-
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.--B P
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SLeadystste60%
0.0 0
200
600
400
800
Stirring rate [rpm]
Figure 4. Effect of stirring rate on the readings of pulsing and steady-stateoxygen electrodes in air-saturated viscous dextran solutions at 25 "C. Table I. Effect of Fouling on the Readings of Pulsing and Steady-State Oxygen Electrodes
oxygen electrode
steady-state pulsing
fouling by fouling by mycelial mycelial newly adhesion adhesion prepared fouling by from a 45-h from a 113-h electrode, antifoam fermentation, fermentation, % agent, % % % 100
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90% with the addition of antifoam agent silicone fluid SAG 471, and there was no effect on the transient current of the pulsing oxygen electrode. Oxygen electrodes from a 45-h cephalosporin C fermentation were taken out of the fermentor and immediately put into air-saturated distilled water. The results show that these electrodes also exhibit a 75-85% drop in the reading of the oxygen electrode, but the transient current remains the same as that of a newly prepared pulsing oxygen electrode. Oxygen electrodes from a 113-hcephalosporin C fermentation were also taken out of the same fermentor to examine the effect of fouling on electrode reading. The membranes of both electrodes were covered by a thin layer of swollen filamentous fragments of Cephalosporium acremonium culture. The reading of this oxygen electrode in airsaturated distilled water decreased 60-70 '% in comparison to that of a newly prepared steady-state oxygen electrode,
0
10
20
30
40
50
Time [hour]
Figure 5. Time course of oxygen tension in the fermentation of cephalosporin C when measured directly by pulsing (-) and steady-state (-) oxygen electrodes and by an off-line steadystate oxygen electrode (w). while the transient current of the pulsing oxygen electrode only showed a slight decrease which is still within 5'% of the actual reading. Continuous Oxygen Measurement during a Cephalosporin C Fermentation. The flow dependence of a steady-state oxygen electrode may be overcomeby constant calibration and by positioning the electrode in a high flow velocity region. But it may still give erroneous readings if the fluid viscosity changes and unavoidable fouling occurs on the electrode membrane surface. For some fermentations, if the initial substrate (or final product) contains bipolymer such as xanthan gum or dextran, the viscosity of the culture broth may vary drastically by the degree of substrate utilization (or product formation) (Cooney, 1985). In a cephalosporin C fermentation, the viscosity of the culture broth is also significantlyinfluenced by the morphological changes of the culture. Initially, the mold grows as filamentous mycelium, which causes an increase in the broth viscosity. At the end of the growth phase, the culture morphology changes again, with the swollen hyphae from arthrospores resulting in a reduction in the viscosity of the broth (Bayer et al., 1989). The time course of dissolved oxygen tension in a cephalosporin C fermentation was monitored by both pulsing and steady-state measurements, and their readings were compared to those measured by an off-line, newly calibrated oxygen electrode. The oxygen tension of the fermentation broth was varied by changing the rate of agitation or aeration. The responses of these two in situ oxygen electrodes were tested by this change of oxygen tension. The result showed that the in situ readings of the steady-state oxygen electrode were almost same as those measured by the pulsing method during the initial 10-15 h of cultivation. However, at the rapid growthstage, the readings of these two electrodes showed considerable deviation due to fouling by mycelia (Figure 5). The addition of an antifoam agent in the fermentation broth will also increase fouling on the electrode membrane. Figures 5 and 6 show the results of measurement of oxygen tension in the fermentation broth with the addition of an antifoam agent at the start-up of fermentation and the growth phase, respectively. If the antifoam agent was added during the growth phase, the time when the readings of these two insitu electrodes became different was delayed until after the addition of the antifoam agent (Figure 6). Zero readings from both electrodes were observed during the rapid growth phase when the dissolved oxygen in the
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Figure 6. Time course of oxygen tension in the fermentation of cephalosporin C when measured directly by pulsing (4 and steady-state (-) oxygen electrodes and by an off-line newly
calibrated steady-state oxygen electrode
(w).
broth was depleted. During this fermentation, both pulsing and steady-state oxygen electrodes showed very good responses to the sudden changes in stirring rate and aeration rate. However,only the readings from the pulsing electrode showed good agreement with those measured by an off-line newly prepared and precalibrated steadystate oxygen electrode. Dissolved oxygen is known to be an important process parameter in most biological processes, and continuous measurement and control of oxygen are always desirable. Control of the oxygen level in these bioprocesses is commonly achieved by a combination of regulating agitation, aeration, and supply of enriched oxygen according to a measured value of oxygen. However, the operating principle of steady-state oxygen measurement makes its readings subject to error due to its dependence on the hydrodynamic conditions of culture broth and on the degree of fouling of the electrode membrane. The error encountered in direct steady-state measurements of dissolved oxygen in some fermentations may be as large as 40% in comparison with the actual dissolved oxygen tension. This is of minor importance in traditional bioprocesses where the dissolved oxygen was controlled to avoid low oxygen tension. But in some industrial bioprocesses, more precise control of the dissolved oxygen level may have physiological implications, and conservative control also increases the cost of agitation and aeration. The advantages of monitoring the controlling dissolved oxygen by the use of the pulsing method with respect to the steady-state method on other fermentations with critical control of oxygen level will be reported in future work.
Conclusion The membrane oxygen electrode usually suffers from long-term signal deterioration due to environmental factors such as changes in hydrodynamic conditions and alteration of membrane oxygen diffusivity due to fouling. The flow dependence of a steady-state oxygen electrode may be overcome by frequent calibration and by placing the electrode in a high flow velocity region (e.g., >30 cm/s). But it may still give erroneous readings if the fluid viscosity changes or fouling occurs on the electrode membrane. For example, even if the stirring rate of a small bioreactor is increased to 600 rpm, the oxygen electrode in a 60% dextran solution still exhibits 35 % reduction in its steady-
state reading compared to that in distilled water. The reading of an oxygen electrode with a membrane fouled by an antifoam agent (silicone fluid SAG 471)also shows a 88-90% reduction. Oxygen electrodes were taken out of the on-going fermentation and immediately put into air-saturated distilled water. They show a 60-85 5% decrease in readings due to biological fouling. The transient current of the pulsing oxygen electrode was found to be quite stable when the pulsing period was controlled at 1s and the rest period lasted longer than 5 min, and it shows minimal interference from hydrodynamic conditions and fouling on the electrode membrane. Dissolved oxygen tension during a cephalosporin C fermentation was monitored by both pulsing and steady-state measurements, and their readings were compared to those measured by an off-line newly calibrated steady-state oxygen electrode. The results showed that the in situ readings of the steady-state oxygen electrode were lower than those measured by the in situ pulsing method during the growth phase and during the stationary phase. The readings from the pulsing electrode showed better agreement with those measured by an off-line precalibrated steady-state oxygen electrode. The error encountered in direct steady-state measurements of dissolved oxygen tension in some fermentations may be as much as 40% off in comparison with the actual dissolved oxygen tension. Notation active surface area of the cathode, cm2 effective thickness of the electrolyte layer, cm effective thickness of the membrane, cm current output from the cathode, A current output from the cathode in the airsaturated distilled water, A effective thickness of the fouling layer, cm diffusivity of oxygen in the electrolytelayer, cm2/s diffusivity of oxygen in the fouling layer, cm2/s diffusivity of oxygen in the membrane, cm2/s diffusivity of oxygen in the bulk solution, cm2/s effective thickness of the boundary layer, cm Faraday constant, 9.65 X lo4 C/g equivalent solubility constant of oxygen in the electrolyte layer, cm3/cm3.atm solubility constant of oxygen in the electrolyte layer, cm3/cm3.atm solubility constant of oxygen in the electrolyte layer, cm3/cm3-atm solubility constant of oxygen in the electrolyte layer, cm3/cm3-atm transient current, A steady current, A partial pressure of oxygen, atm time, s volumetric flow rate of air per volume of fermentation broth, cm3/cm3.s Acknowledgment The authors acknowledge the partial financial assistance of the National Science Foundation for this work. We also thank Miss Tuo-ju Liu for technical assistance in this study. Literature Cited Bayer, T.; Zhou, W.; Holzhauer, K.; Schugerl, K. Investigations of Cephalosporin C production in an airlift tower loop reactor. Appl. Microbiol. Biotechnol. 1989, 30,26-33.
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Bessette, R. R.; Harwood, D. F.; Brainard, E. C., I1 Performance characteristics and calibration of the EMDECO pulsed dissolvedoxygenType 1125system. IEEE J.OceanicEng. 1988, 13 (3), 140-143.
Buehler, H.W.; Bucher, R. Applications of electrochemical electrodes. In Sensors in Bioprocess Control; Twork, J. V.; Yacynych, A. M., Eds.; Marcel Dekker: New York, 1990; pp 142-154.
Clark, L. c., Jr.; Wolf, R. G. D.; Taylor, Z. Continuous recording of blood oxygen tensions by polarography. J. Appl. Physiol. 1953,6,189-193.
Cooney, C. L. Growth of microorganisms. In Biotechnology; Rehm, H. J., Reed, G., Eds.; Verlag Chemie: Weinheim, Germany, 1985;Vol. 1, pp 96-97. Fatt, I. The Polarographic Oxygen Electrode; CRC Press: Cleveland, OH, 1976;pp 9-61. Haddad, I. A. Method of measuring oxygen using a membrane covered polarographic electrode. U.S.Patent 3,718,562,1973. Hitchman, M. L. Measurement of Dissolved Oxygen; WileyInterscience; New York, 1978;pp 59-129. Kuessner, A. Indirect application of a membrane-covered electrochemical Clark cell electrode for the determination of molecular oxygen in gaseous, liquid or solid samples. Phys. E.: Sci. Instrum. 1987,20,224-230. Langdon, C. Dissolved oxygen monitoring system using a pulsed electrode: design, performance, and evaluation. Deep-sea Res. 1984,31 (ll),1357-1367. Lee,Y. H.; Tsao,G. T. Dissolved oxygenelectrodes. In Advances in Biochemical Engineering; Ghose, T. K., Fiechter, A., Blakebrough, N., Eds.; Springer-Verlag: Berlin, 1979;Vol. 13, pp 38-83. Lilley,M. D.; Story,J. B.; Raible, R. W. The chronoamperometric determination of dissolved oxygen usingmembrane electrodea. J. Electroanal. Chem. 1969,23,425-429. Linek, V. Dynamicmeasurement of the volumetric mass transfer coefficientin agitated vessels: effect of the start-up peroid on
the response of an oxygenelectrode. Biotechnol.Bioeng. 1977, 16,983-1008.
Mancy, K. H.In situ measurement of dissolved oxygen by pulse and steady state voltammetric membrane electrode system. In Chemistry and Physics of Aqueous Gas Solutions;Adams, W. A., Ed.; The Electrochemical Society: Princeton, NJ, 1975; pp 281-289. Mancy, K. H.; Okun, D. A.; Reilley,C. N. A galvanic cell oxygen analyzer. J. Electroanal. Chem. 1962,4,65-92. Myland, J. C.; Oldham, K. B. Membrane-covered oxygen electrode. An exact treatment of the switch-on transient. J. Electrochem. SOC.: Electrochem. Sci. Technol. 1984,131 (8), 18161823.
Shen, Y. Q.;Wolfe, S.; Demain, A. L. Levels of isopenicillin N synthetase and deawtoxywphaloeporinC synthetasein Cephalosporium acremonium producing high and low levels of cephalosporin C. BiolTechnology 1986,4,61-64. Short, D. L.;Shell, G. S. G. Pulsing amperometric oxygen electrodes: earlier techniques evaluated and a technique implemented to cancel capacitive charge. J. Phys. E: Sci. Instrum. 1986,18,79-87. Smith, A. Cephalosporins. In Comprehensive Biotechnology; Moo-Young,M., Ed.; Pergamon Press: Oxford, 1985;Vol. 3, pp 177-178. Thomas, L. C.; Chamberlin, G. J. The determinationof dissolved oxygen. In Colorimetric Chemical Analytical Methods; The Tintometer Ltd.: England, 1967;pp 291-303. Wang, H. Y.; Li, X. M. Transient measurement of diseolved oxygen using membrane electrodes. Biosensor 1989,4,273285.
Wilson, A. L. The Chemical Analysis of Water; The Chemical Society: London, 1974;p 124. Accepted October 12, 1992.
