- Email: [email protected]
Development of a dissolved hydrogen sensor and its application to evaluation of hydrogen mass transfer
Jotrm~AL OF FERMENTATIONAND BIOENGINEERING VOI. 76, NO. 2, 148-150. 1993
Development of a Dissolved Hydrogen Sensor and Its Application to Evaluation of Hydrogen Mass Transfer T O S H I H I R O TAKESHITA, l KENJI TANAKA, 1 AYAAKI ISHIZAKI, 1. AND PETER F. STANBURY 2
Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, 46-09 Hakozaki, Higashi-ku, Fukuoka 812, Japan 1 and Division of Bioscience, University of Hertfordshire, Hatfield, Herts, A L IO 9AB, United Kingdom 2 Received 8 February 1993/Accepted 27 April 1993
The partial pressure of dissolved hydrogen was measured by a dissolved hydrogen sensor constructed by modifying a Clark-type dissolved oxygen probe. Using this sensor, the overall volumetric coefficient of hydrogen mass transfer, (KLa)H2, was determined in the culture system and compared with that of oxygen, (KLa)o2~ol.. Good straight line relationships were demonstrated between (i) ln(K~a)o2bloi, and In(Ku=)o~==. (K~a for oxygen determined by sulphite oxidation) and (ii) In(Ku~)o2bio, and In(KLa)H2.
We have developed a recycled-gas closed-circuit culture system for the production of PHB from high cell density cultures o f A . eutrophus ATCC 17697 T growing on carbon dioxide and hydrogen under aerobic conditions (1, 2). According to the stoichiometry of this organism (1, 2) the ratio of H E : 02 : C O 2 required for cell growth is about 21.36:6.21:4.09 and that for PHB accumulation is 33 : 12 : 4. Thus, to determine the possible gas composition without the detonation range it is necessary to study the kinetics of the substrate gasses in the culture system. The mass transfer kinetics of oxygen and carbon dioxide have been well documented (3, 4). However, although there are many reports measuring dissolved hydrogen concentration (5-13), very little data are available on the determination of the mass transfer kinetics of hydrogen in a fermentor. In this paper the development of a dissolved hydrogen sensor is reported, and the kinetics of hydrogen mass transfer in the fermentation system are compared with that of oxygen. The strain, medium preparation and analytical methods were as previously reported (1, 2). Figure 1 shows the dimensions of the fermentation vessel used for mass transfer analyses. The vessel is equipped with pH, dissolved oxygen (DO) and dissolved hydrogen (DH) sensors, whose lengths in the vessel are 100 m m for the pH and DH electrodes and 90 m m for the DO electrode. The DH sensor shown in Fig. 2 was developed by modifying a Clark-type DO sensor, DG-SP, 134 m m long and 10mm in diameter (Biott Co., Ltd., Tokyo), using a very similar method to that reported by previous workers (11, 12). The sensor consisted of a silver/silver chloride cathode and a platinum/platinum black anode with 1 M KC1 (pH 1.0 with HC1) as the internal electrolyte. To coat the spiral Ag electrode with AgC1, the probe was filled with a 1 M KCI solution and electrolyzed at DC 10mA for 1 d using a platinum plate as a counter electrode. The solution was then discarded and the inside of the probe rinsed with 1 M KCI. Similarly, to coat the plane disc Pt electrode with platinum black (Pt black) the tip of the probe was dipped into a 3% hydrogen hexachloroplatinate (IV), hexahydrate solution without lead acetate and was
electrolyzed at DC 5.0V for 1.5 min using a platinum plate as a counter electrode. The probe was then rinsed with 1 M KCI. The tip of the sensor was covered with a 2.54Fm thick polytetrafluoroethylene membrane which was fixed to the sensor with 3 O-rings (i.d. 3.7 m m and o.d. 7.5 ram) and silicone lubricant SH-103 (Toray Dow Coming, Tokyo) as a sealing material. Finally, the sensor was filled with 1 M KCI electrolyte, the p H of which had been adjusted to 1.0 with HCI to ensure that the sensor would not be affected by pH changes. The electrical circuit was designed to generate a polarizing potential of DC 550 mV between the Pt-Pt black anode (sensing electrode) and the Ag-AgC1 cathode (counter electrode) to achieve hydrogen oxidation (9, 11). The polarizing potential was continuously monitored by a digital multi tester, model 7532-01 (Yokogawa Instruments Co.,
Gas out
Gas in
-]r--II
•
0
¢' 3.0-')
l_r I~
40,0 49.5
FIG. 1. Dimensions o f the vessel used for KLa determination. Unit: millimeters.
* Corresponding author. 148
NOTES
VoL. 76, 1993
149
TABLE 1. Volumetric mass transfer coefficients (h -l)
(KLa)o~sut. (KLa)O2biol. (KLa)H2
1lO0 257 385 628
Agitation (rpm) 1400 319 558 847
1700 417 716 1430
Vent hole Electrolyte M KCI, pH 1.0 with HCI)
CathodelAg-~
Glass tubic
to the set value. The change in the dissolved hydrogen partial pressure was recorded by the DH sensor. Because the hydrogen consumption rate was constant throughout the gassing out stage the following relationship describes the hydrogen consumption rate during this stage: RH2 : HH2" (PLB -- PL)" t - l
(1)
At steady state dissolved hydrogen concentration, the hydrogen consumption rate equals the hydrogen transfer rate into solution, thus: RIIE : (KLa)n2"/-/HE" (PG -- PEa)
Thus, the overall volumetric coefficient of hydrogen mass transfer in the biological system, (KLa)H2, can be calculated from the following equation:
O-ri Anode(Pt-Pt black)
(KLa)n2 = (PEa -- PL)" {(PG -- PLB)" t} -1
PTFE membrame
FIG. 2. Cross-sectional view of the dissolved hydrogen sensor.
Ltd.,Tokyo) with a high input impedance of more than 10 Mr). The output current from the sensor was converted into a voltage signal by a 101.3 t) resistor and the voltage value recorded on a recorder, PRP-5021 (Toa Electronics Ltd., Tokyo) with a high input impedance of more than 1 Mr2. The partial pressure of dissolved oxygen was measured with a galvanic oxygen sensor, 10AN-S (Biott Co. Ltd., Tokyo), and a dissolved oxygen meter, AI-1007 (Biott Co. Ltd., Tokyo). The output voltage from the meter was recorded on the same PRP-5021 recorder used for the hydrogen sensor. The overall volumetric coefficient of hydrogen mass transfer was determined during the autotrophic batch culture of A. eutrophus ATCC 17697 T. When the dry cell weight reached 8 . 0 g . l -I, the gas supply was suspended and the agitation speed lowered to 250 rpm and then the gas supply was resumed and the agitation speed returned 30
i
20 10
0~ 0.0
(2)
I
I
I
i
0.2 0.4 0.6 0.8 H 2 partial pressure in distilled water [atm]
1.0
FIG. 3. Calibration of the assembled dissolved hydrogen sensor.
(3)
The overall volumetric coefficient of oxygen mass transfer in the fermentor was also determined by using the sulphite oxidation method, (KLt/)OEsul., and by the dynamic gassing out and steady state method, (KLg)O2biol.. The D H sensor was calibrated using hydrogen dissolved in distilled water. Output currents from the DH sensor at various partial pressures of hydrogen are shown in Fig. 3. When dissolved hydrogen was removed from distilled water by sparging with nitrogen, the output current from the D H sensor was 205 nA. The output current generated by the D H sensor under hydrogen saturated conditions was 28.4/aA. The correlation coefficient of the linear relationship in Fig. 3 is 0.999. Although sulphite compounds are known to affect the current output of DH sensors (9) it was confirmed that none of the ingredients used in the culture medium interfered with the probe response. (KLt/)H2, (gLa)o2sul" and (KLa)O2biol. were determined at agitation speeds of 1100, 1400 and 1700 rpm. The results of these determinations are shown in Table I. For oxygen, the KLa value determined using the dynamic gassing out and steady state method, (KLa)O2bioLwas larger than the value obtained using the sulphite oxidation method, (KLa)O2~uL, a phenomenon also reported by Hsieh et al. (14). The (KLa)H2value was higher than that of (KLa)o2bioL, supporting similar data by Kodama et al. (5) generated from microorganism-free media. The relationship between the overall volumetric coefficients for oxygen determined by the sulphite method and the dynamic gassing out and steady state method was investigated in the natural logarithm plot of the two fact o r s . The relationship between the natural logarithms of the determined (KLa)O2bioL and (KLa)o2suL is expressed in equation (4). Also, the relationship between the natural logarithms of (KLa)iq2 and (KLa)O2biol. is expressed in equation (5). (KLa)O2biol. = 0.347(KLa)o2su1.1"27
(4)
(gLa)H 2 = 0.280(KLa)O2biol. 1"29
(5)
150
TAKESHITA ET AL.
NOMENCLATURE :Henry's constant for hydrogen, mol./-1. kPa-l : o v e r a l l v o l u m e t r i c coefficient o f h y d r o g e n (KLa)H 2 mass t r a n s f e r in b i o l o g i c a l system, h -~ (KLa)O2biol. : overall v o l u m e t r i c coefficient o f o x y g e n mass t r a n s f e r in b i o l o g i c a l system, h -~ (KLa)o2suL : overall v o l u m e t r i c coefficient o f o x y g e n mass t r a n s f e r in sulfite o x i d a t i o n system, h -1 : partial p r e s s u r e o f h y d r o g e n in e x h a u s t gas, Po kPa PL : partial p r e s s u r e o f dissolved h y d r o g e n at gas on, kPa : p a r t i a l pressure o f dissolved h y d r o g e n at gas PLB off, k P a : h y d r o g e n c o n s u m p t i o n rate, m o l . 1-1- h RH2 t : time, h HH 2
The authors express their sincere appreciation to Y. Ishikawa of Biott Co. Ltd. for providing the electrode materials and technical support. REFERENCES 1. Ishizaki, A. and Tanaka, K.: Production of poly-/%hydroxybutyric acid from carbon dioxide by Alcaligenes eutrophus ATCC 17697T. J. Ferment. Bioeng., 71, 254-257 (1991). 2. Ishizaki, A. and Tanaka, K.: Batch culture of Alcaligenes eutrophus ATCC 17697T using recycled gas closed circuit culture system. J. Ferment. Bioeng., 69, 170-174 (1990). 3. Ishizaki, A., Shibai, H., Hirose, Y., and Shiro, T.: Estimation of aeration and agitation conditions in respect to oxygen supply and ventilation. Agr. Biol. Chem., 37, 107-113 (1973).
J. FERMENT. BIOENG., 4. lshizaki, A. and Hirose, Y.: Kinetics of carbon dioxide evolution in agitation system. Agr. Biol. Chem., 37, 1295-1305 (1973). 5. Kodama, T., Goto, E., and Mlnoda, Y.: Determination of dissolved hydrogen concentration and [KLa]~z in submerged culture vessel. AgE Biol. Chem., 40, 2373-2377 (1976). 6. Siegel, R. S. and Ollis, D. F.: Kinetics of growth of the hydrogenoxidizing bacterium Alcaligenes eutrophus (ATCC 17707) in chemostat culture. Biotechnol. Bioeng., 26, 764-770 (1984). 7. Heinzle, E. and Laffety, R.M.: Continuous mass spectrometric measurement of dissolved H2, 02, and CO2 during chemolithoautotrophic growth of Alcaligenes eutrophus strain H 16. Eur. J. Appl. Microbial Biotechnol., 11, 17-22 (1980). 8. Panss, A., Samson, R., Guiot, S., and Beauchemin, C.: Continuous measurement of dissolved H2 in an anaerobic reactor using a new hydrogen/air fuel cell detector. Biotechnol. Bioeng., 35, 492-501 (1990). 9. Kuroda, K., Siiveira, R.G., Nlshio, N., Sunahara, H., and Nagai, S.: Measurement of dissolved hydrogen in an anaerobic digestion process by a membrane-covered electrode. J. Ferment. Bioeng., 71, 418-423 (1991). 10. Sweet, W.J., Houchlns, J.P., Rosen, P.R., and Aria, D.J.: Polarographic measurement of H2 in aqueous solution. Anal. Biochem., 107, 337-340 (1980). 11. Niedrach, L. W. and Stoddard, W.H.: Continuous voltametric monitoring of hydrogen and oxygen in water. Anal. Chem., 54, 1651-1654 (1982). 12. Kurosawa, K. and Hagihara, B.: Measurement of oxygen and hydrogen by polarographic method. Taisya (in Japanese), 23, 555-561 (1986). 13. Ohi, K., Nishimura, T., Okazaki, M., and Miura, Y.: Determination of dissolved hydrogen concentration during cultivation and assimilation of gaseous substrates by Alcaligenes hydrogenophilus. J. Ferment. Technol., 57, 203-209 (1979). 14. Hsieh, D. P. H., Silver, R. S., and Mateles, R. I.: Use of the glucose oxidase system to measure oxygen transfer rates. Biotechnol. Bioeng., 11, 1-18 (1969).
Development of a Dissolved Hydrogen Sensor and Its Application to Evaluation of Hydrogen Mass Transfer T O S H I H I R O TAKESHITA, l KENJI TANAKA, 1 AYAAKI ISHIZAKI, 1. AND PETER F. STANBURY 2
Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, 46-09 Hakozaki, Higashi-ku, Fukuoka 812, Japan 1 and Division of Bioscience, University of Hertfordshire, Hatfield, Herts, A L IO 9AB, United Kingdom 2 Received 8 February 1993/Accepted 27 April 1993
The partial pressure of dissolved hydrogen was measured by a dissolved hydrogen sensor constructed by modifying a Clark-type dissolved oxygen probe. Using this sensor, the overall volumetric coefficient of hydrogen mass transfer, (KLa)H2, was determined in the culture system and compared with that of oxygen, (KLa)o2~ol.. Good straight line relationships were demonstrated between (i) ln(K~a)o2bloi, and In(Ku=)o~==. (K~a for oxygen determined by sulphite oxidation) and (ii) In(Ku~)o2bio, and In(KLa)H2.
We have developed a recycled-gas closed-circuit culture system for the production of PHB from high cell density cultures o f A . eutrophus ATCC 17697 T growing on carbon dioxide and hydrogen under aerobic conditions (1, 2). According to the stoichiometry of this organism (1, 2) the ratio of H E : 02 : C O 2 required for cell growth is about 21.36:6.21:4.09 and that for PHB accumulation is 33 : 12 : 4. Thus, to determine the possible gas composition without the detonation range it is necessary to study the kinetics of the substrate gasses in the culture system. The mass transfer kinetics of oxygen and carbon dioxide have been well documented (3, 4). However, although there are many reports measuring dissolved hydrogen concentration (5-13), very little data are available on the determination of the mass transfer kinetics of hydrogen in a fermentor. In this paper the development of a dissolved hydrogen sensor is reported, and the kinetics of hydrogen mass transfer in the fermentation system are compared with that of oxygen. The strain, medium preparation and analytical methods were as previously reported (1, 2). Figure 1 shows the dimensions of the fermentation vessel used for mass transfer analyses. The vessel is equipped with pH, dissolved oxygen (DO) and dissolved hydrogen (DH) sensors, whose lengths in the vessel are 100 m m for the pH and DH electrodes and 90 m m for the DO electrode. The DH sensor shown in Fig. 2 was developed by modifying a Clark-type DO sensor, DG-SP, 134 m m long and 10mm in diameter (Biott Co., Ltd., Tokyo), using a very similar method to that reported by previous workers (11, 12). The sensor consisted of a silver/silver chloride cathode and a platinum/platinum black anode with 1 M KC1 (pH 1.0 with HC1) as the internal electrolyte. To coat the spiral Ag electrode with AgC1, the probe was filled with a 1 M KCI solution and electrolyzed at DC 10mA for 1 d using a platinum plate as a counter electrode. The solution was then discarded and the inside of the probe rinsed with 1 M KCI. Similarly, to coat the plane disc Pt electrode with platinum black (Pt black) the tip of the probe was dipped into a 3% hydrogen hexachloroplatinate (IV), hexahydrate solution without lead acetate and was
electrolyzed at DC 5.0V for 1.5 min using a platinum plate as a counter electrode. The probe was then rinsed with 1 M KCI. The tip of the sensor was covered with a 2.54Fm thick polytetrafluoroethylene membrane which was fixed to the sensor with 3 O-rings (i.d. 3.7 m m and o.d. 7.5 ram) and silicone lubricant SH-103 (Toray Dow Coming, Tokyo) as a sealing material. Finally, the sensor was filled with 1 M KCI electrolyte, the p H of which had been adjusted to 1.0 with HCI to ensure that the sensor would not be affected by pH changes. The electrical circuit was designed to generate a polarizing potential of DC 550 mV between the Pt-Pt black anode (sensing electrode) and the Ag-AgC1 cathode (counter electrode) to achieve hydrogen oxidation (9, 11). The polarizing potential was continuously monitored by a digital multi tester, model 7532-01 (Yokogawa Instruments Co.,
Gas out
Gas in
-]r--II
•
0
¢' 3.0-')
l_r I~
40,0 49.5
FIG. 1. Dimensions o f the vessel used for KLa determination. Unit: millimeters.
* Corresponding author. 148
NOTES
VoL. 76, 1993
149
TABLE 1. Volumetric mass transfer coefficients (h -l)
(KLa)o~sut. (KLa)O2biol. (KLa)H2
1lO0 257 385 628
Agitation (rpm) 1400 319 558 847
1700 417 716 1430
Vent hole Electrolyte M KCI, pH 1.0 with HCI)
CathodelAg-~
Glass tubic
to the set value. The change in the dissolved hydrogen partial pressure was recorded by the DH sensor. Because the hydrogen consumption rate was constant throughout the gassing out stage the following relationship describes the hydrogen consumption rate during this stage: RH2 : HH2" (PLB -- PL)" t - l
(1)
At steady state dissolved hydrogen concentration, the hydrogen consumption rate equals the hydrogen transfer rate into solution, thus: RIIE : (KLa)n2"/-/HE" (PG -- PEa)
Thus, the overall volumetric coefficient of hydrogen mass transfer in the biological system, (KLa)H2, can be calculated from the following equation:
O-ri Anode(Pt-Pt black)
(KLa)n2 = (PEa -- PL)" {(PG -- PLB)" t} -1
PTFE membrame
FIG. 2. Cross-sectional view of the dissolved hydrogen sensor.
Ltd.,Tokyo) with a high input impedance of more than 10 Mr). The output current from the sensor was converted into a voltage signal by a 101.3 t) resistor and the voltage value recorded on a recorder, PRP-5021 (Toa Electronics Ltd., Tokyo) with a high input impedance of more than 1 Mr2. The partial pressure of dissolved oxygen was measured with a galvanic oxygen sensor, 10AN-S (Biott Co. Ltd., Tokyo), and a dissolved oxygen meter, AI-1007 (Biott Co. Ltd., Tokyo). The output voltage from the meter was recorded on the same PRP-5021 recorder used for the hydrogen sensor. The overall volumetric coefficient of hydrogen mass transfer was determined during the autotrophic batch culture of A. eutrophus ATCC 17697 T. When the dry cell weight reached 8 . 0 g . l -I, the gas supply was suspended and the agitation speed lowered to 250 rpm and then the gas supply was resumed and the agitation speed returned 30
i
20 10
0~ 0.0
(2)
I
I
I
i
0.2 0.4 0.6 0.8 H 2 partial pressure in distilled water [atm]
1.0
FIG. 3. Calibration of the assembled dissolved hydrogen sensor.
(3)
The overall volumetric coefficient of oxygen mass transfer in the fermentor was also determined by using the sulphite oxidation method, (KLt/)OEsul., and by the dynamic gassing out and steady state method, (KLg)O2biol.. The D H sensor was calibrated using hydrogen dissolved in distilled water. Output currents from the DH sensor at various partial pressures of hydrogen are shown in Fig. 3. When dissolved hydrogen was removed from distilled water by sparging with nitrogen, the output current from the D H sensor was 205 nA. The output current generated by the D H sensor under hydrogen saturated conditions was 28.4/aA. The correlation coefficient of the linear relationship in Fig. 3 is 0.999. Although sulphite compounds are known to affect the current output of DH sensors (9) it was confirmed that none of the ingredients used in the culture medium interfered with the probe response. (KLt/)H2, (gLa)o2sul" and (KLa)O2biol. were determined at agitation speeds of 1100, 1400 and 1700 rpm. The results of these determinations are shown in Table I. For oxygen, the KLa value determined using the dynamic gassing out and steady state method, (KLa)O2bioLwas larger than the value obtained using the sulphite oxidation method, (KLa)O2~uL, a phenomenon also reported by Hsieh et al. (14). The (KLa)H2value was higher than that of (KLa)o2bioL, supporting similar data by Kodama et al. (5) generated from microorganism-free media. The relationship between the overall volumetric coefficients for oxygen determined by the sulphite method and the dynamic gassing out and steady state method was investigated in the natural logarithm plot of the two fact o r s . The relationship between the natural logarithms of the determined (KLa)O2bioL and (KLa)o2suL is expressed in equation (4). Also, the relationship between the natural logarithms of (KLa)iq2 and (KLa)O2biol. is expressed in equation (5). (KLa)O2biol. = 0.347(KLa)o2su1.1"27
(4)
(gLa)H 2 = 0.280(KLa)O2biol. 1"29
(5)
150
TAKESHITA ET AL.
NOMENCLATURE :Henry's constant for hydrogen, mol./-1. kPa-l : o v e r a l l v o l u m e t r i c coefficient o f h y d r o g e n (KLa)H 2 mass t r a n s f e r in b i o l o g i c a l system, h -~ (KLa)O2biol. : overall v o l u m e t r i c coefficient o f o x y g e n mass t r a n s f e r in b i o l o g i c a l system, h -~ (KLa)o2suL : overall v o l u m e t r i c coefficient o f o x y g e n mass t r a n s f e r in sulfite o x i d a t i o n system, h -1 : partial p r e s s u r e o f h y d r o g e n in e x h a u s t gas, Po kPa PL : partial p r e s s u r e o f dissolved h y d r o g e n at gas on, kPa : p a r t i a l pressure o f dissolved h y d r o g e n at gas PLB off, k P a : h y d r o g e n c o n s u m p t i o n rate, m o l . 1-1- h RH2 t : time, h HH 2
The authors express their sincere appreciation to Y. Ishikawa of Biott Co. Ltd. for providing the electrode materials and technical support. REFERENCES 1. Ishizaki, A. and Tanaka, K.: Production of poly-/%hydroxybutyric acid from carbon dioxide by Alcaligenes eutrophus ATCC 17697T. J. Ferment. Bioeng., 71, 254-257 (1991). 2. Ishizaki, A. and Tanaka, K.: Batch culture of Alcaligenes eutrophus ATCC 17697T using recycled gas closed circuit culture system. J. Ferment. Bioeng., 69, 170-174 (1990). 3. Ishizaki, A., Shibai, H., Hirose, Y., and Shiro, T.: Estimation of aeration and agitation conditions in respect to oxygen supply and ventilation. Agr. Biol. Chem., 37, 107-113 (1973).
J. FERMENT. BIOENG., 4. lshizaki, A. and Hirose, Y.: Kinetics of carbon dioxide evolution in agitation system. Agr. Biol. Chem., 37, 1295-1305 (1973). 5. Kodama, T., Goto, E., and Mlnoda, Y.: Determination of dissolved hydrogen concentration and [KLa]~z in submerged culture vessel. AgE Biol. Chem., 40, 2373-2377 (1976). 6. Siegel, R. S. and Ollis, D. F.: Kinetics of growth of the hydrogenoxidizing bacterium Alcaligenes eutrophus (ATCC 17707) in chemostat culture. Biotechnol. Bioeng., 26, 764-770 (1984). 7. Heinzle, E. and Laffety, R.M.: Continuous mass spectrometric measurement of dissolved H2, 02, and CO2 during chemolithoautotrophic growth of Alcaligenes eutrophus strain H 16. Eur. J. Appl. Microbial Biotechnol., 11, 17-22 (1980). 8. Panss, A., Samson, R., Guiot, S., and Beauchemin, C.: Continuous measurement of dissolved H2 in an anaerobic reactor using a new hydrogen/air fuel cell detector. Biotechnol. Bioeng., 35, 492-501 (1990). 9. Kuroda, K., Siiveira, R.G., Nlshio, N., Sunahara, H., and Nagai, S.: Measurement of dissolved hydrogen in an anaerobic digestion process by a membrane-covered electrode. J. Ferment. Bioeng., 71, 418-423 (1991). 10. Sweet, W.J., Houchlns, J.P., Rosen, P.R., and Aria, D.J.: Polarographic measurement of H2 in aqueous solution. Anal. Biochem., 107, 337-340 (1980). 11. Niedrach, L. W. and Stoddard, W.H.: Continuous voltametric monitoring of hydrogen and oxygen in water. Anal. Chem., 54, 1651-1654 (1982). 12. Kurosawa, K. and Hagihara, B.: Measurement of oxygen and hydrogen by polarographic method. Taisya (in Japanese), 23, 555-561 (1986). 13. Ohi, K., Nishimura, T., Okazaki, M., and Miura, Y.: Determination of dissolved hydrogen concentration during cultivation and assimilation of gaseous substrates by Alcaligenes hydrogenophilus. J. Ferment. Technol., 57, 203-209 (1979). 14. Hsieh, D. P. H., Silver, R. S., and Mateles, R. I.: Use of the glucose oxidase system to measure oxygen transfer rates. Biotechnol. Bioeng., 11, 1-18 (1969).