measurement of dissolved hydrogen in an anaerobic digestion process by a membrane-covered electrode

JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 71, No. 6, 418-423. 1991

Measurement of Dissolved Hydrogen in an Anaerobic Digestion Process by a Membrane-Covered Electrode

KENJI KURODA, 1 ROBERTO G A I G E R SILVEIRA, 1 N A O M I C H I NISHIO, l H I R O S H I S U N A H A R A , 2 AND SHIRO N A G A P *

Department of Fermentation Technology, 1 Department of Environmental Science, 2 Faculty of Engineering, Hiroshima University, 4-1, Kagamiyama l-chome, Higashi-Hiroshima 724, Japan

Received 12 December 1990/Accepted 8 March 1991

Dissolved hydrogen in an anaerobic digestion process was continuously measured by a voltammetric membrane electrode which consisted of a Pt-Pt black and Ag-AgCI covered FEP membrane with 0.1 M KCI and 0.1 M HCI. This sensor showed high reliability and sensitivity (i.e., detection limit: 50 nM) in distilled water. The sensor was not affected by several compounds in the anaerobic digestion media (e.g., inorganic salts, acetate, and propionate) except for sulfide. The indication in a sample containing 1.56 mM sulfide corresponded to that of 0.26/~M dissolved hydrogen. The sensor was also applied to measure the dissolved hydrogen in a laboratory-scale anaerobic reactor, and the dissolved hydrogen was continuously monitored for 565-h. The sensor was calibrated every 120-h, and the output signal was very stable during this period. The dissolved hydrogen concentration ranged from 0.5 to 3/~M, and H2 partial pressure from 2 to 7 Pa in the gas phase. A good correlation (r = 0.85) between theoretical values calculated with H2 partial pressure and the output signals was recognized. The actual dissolved hydrogen concentration was about 60-fold higher than the theoretical values calculated with H2 partial pressure.

In methanogenic ecosystems it is a well-known fact that accumulated hydrogen strongly inhibits the degradat ion of volatile fatty acids, such as propionic and butyric acids, resulting in a consequent deter iorat ion of the normal oper- a t ion (1-3). It is therefore significant to measure the dissolved hydrogen concentra t ion in anaerobic digestion processes.

The very low solubil i ty of hydrogen in water, i.e., 67 nM for a hydrogen part ia l pressure of 10 Pa at 37°C, makes quantif icat ion delicate. H2 part ia l pressure has been determined by gas chromatograph methods so far (4), and the dissolved hydrogen concentra t ion has been calculated by the Bunsen absorp t ion coefficient or Henry ' s constant (5, 6) under the assumpt ion that the hydrogen transfer rate between the gas and liquid phases is not limited. However , as the culture bro th of anaerobic digestions consists of a com- plex physico-chemical construct ion with respect to hydrogen solubili ty, it is uncertain whether this method is cor- rect or not. Some other techniques have also been developed for measuring dissolved hydrogen directly through Teflon tubing connected to a gas chromatograph equipped with a mercury-mercuric oxide detector, and by membrane-inlet quadrupole mass spectrometry (7-10), but their appl icabi l i ty to anaerobic digestions is still l imited due to their sophist ication, high cost, detection limits and interference f rom other solutes.

Recently, Pauss et al. have repor ted a new technique using a hydrogen /a i r fuel cell detector (11) which was able to measure dissolved hydrogen with long term stable opera- t ion (40 d) in an anaerobic reactor.

In this study, we used a membrane-covered electrode (DH2 sensor) to measure dissolved hydrogen directly in an anaerobic digestion process. Al though DHz-sensors have al ready been developed for measuring dissolved hydrogen (12, 13), these have been tested only in pure water and no

* Corresponding author.

measurement has been performed in a fermentat ion broth. Here, we apply this sensor to an anaerobic digestion reactor, and evaluate its sensitivity, selectivity, and long-term stability. In addi t ion, we discuss the correlat ion between the output signal of the DH2-sensor and H2 part ial pressure determined by a gas chromatograph method.

M A T E R I A L S AND M E T H O D S

Dissolved hydrogen electrode The dissolved hydrogen detector was provided by T O A Electronics Ltd. (Model D H D I - I , Tokyo) , and consisted of a dissolved hydrogen electrode, and an amplified and applied voltage source using a polarographic circuit. This detector was developed for water quali ty control in nuclear plants and for the corrosion testing of metal, and is now in practical use. According to the supplier 's technical data, the indicat ion of the sensor is little affected by the hydrogen gas flow rate, but is sensitive to the liquid circulation rate. Therefore, it is necessary to mainta in a constant flow rate of the sample solut ion during measurement . In addi t ion, excellent linear- ity between the indications of the DH2 sensor and the dissolved hydrogen concentrat ion calculated with H 2 part ia l pressure was obtained within the range from 0 to 10 mg/! in pure water. When N2 and H2 gases were directly passed by turns to the DH2-electrode, high responsiveness (90~0 response within 30 s) and reproducibi l i ty (l°J0 variation) were obtained.

The dissolved hydrogen electrode (HE-5321, TOA) was covered with a FEP (Fluorinated Ethylene-Propylene) membrane with an internal electrolyte of 0.1 M KC1 and 0.1 M HC1. The anode was formed by p la t inum-pla t inum black and the cathode was silver-silver chloride. The anode (working electrode) was polarized at + 0.55 V applied potent ial to the counter electrode. Both electrodes were filled with the internal electrolyte and then covered with FEP membrane 25/~m in thickness.

418

VoL. 71, 1991 DISSOLVED H2 MEASUREMENT IN ANAEROBIC DIGESTION 419

5 6

13

4 9

121 0

FIG. 1. Schematic diagram of calibration system. (1) 1 l bottle (distilled water or medium: 0.8/); (2) DH2 electrode; (3) flow cell; (4) circulation pump; (5) amplifier; (6) recorder; (7) gas cylinder; (8) pressure regulator; (9) flow meter; (10) glass-ball filter; (11) gas outlet; (12) magnetic stirrer; (13) thermo-controller (37°C).

The principle of measurement is as follows: a dissolved hydrogen sample solut ion diffuses into the electrodes through the selective permeable membrane, and the dissolved hydrogen is oxidized at the appl ied voltage of +0.55 V vs. Ag-AgC1. An oxidat ion current p ropor t ion- ally corresponding to the dissolved hydrogen concentra- t ion is recorded automat ical ly .

Calibration of dissolved hydrogen In order to cal ibrate the output signal corresponding to H 2 at the gas phase, cal ibrat ion was carried out by N2 and H 2 gases. The reading of the DH2 detector by using N 2 gas (99.990/00) was set to 0% by electrical adjus tment after the indicat ion was stabilized. After this, H 2 gas (99.99~0) was used, and the indicat ion was set to 100% by another electrical adjus tment after a constant value was obtained. The cal ibrat ion was carried out about every month to check the stabili ty before use.

Af ter the cal ibrat ion, dissolved hydrogen in the liquid phase was measured by using distilled water, bubbled N2 or H2 gas. As shown in Fig. 1, an apparatus was constructed for the measurements . The DH2-electrode was inserted in the flow cell (3 in Fig. 1) and the sample solut ion was circulated f rom the reserve tank (1 in Fig. 1) to the flow cell by a circulation pump (4 in Fig. 1). The volume for the flow of the sample solution in the flow cell is 3.6 ml by sub- t ract ion from the total volume of the flow cell (inner diameter, 31 .Smm; height, 41 mm) of the DH2-electrode volume (diameter, 30 ram; height, 39 mm), and the flow rate on the electrode surface was obl igator i ly set at 0 . 01 / /min due to the motive force o f the pump; hence, the indicat ion of the DH2 detector was 93 ~ compared to the sa tura t ion level (680/aM). N2 gas was bubbled for 30 min in distilled water, and the reading of the D H 2 detector corresponded to 0.15/aM of dissolved hydrogen, but this value was considered to correspond to a residual current. When H2 gas was bubbled for 30 min, a stable output signal was obta ined by bubbl ing H2 for 10min. As the full scale o f detector was 630/aM and there is no suitable method for the chemical analysis of dissolved hydrogen in solution, a well-known and calculated value for the saturated concentration of dissolved hydrogen was used as a ref- erence, that is, the value of 680/~M. The concentrat ions of dissolved hydrogen obta ined in this experiment were cal ibrated as values corresponding to 680/aM of dissolved hydrogen.

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_Jl FIG. 2. Schematic diagram of methanogenic reactor equipped

with DH2-detector. (1) 2.3 l methanogenic reactor (medium: 1.6/); (2) DH2-electrode; (3) flow cell; (4) circulation pump; (5) amplifier; (6) pH electrode; (7) pH controller; (8) recorder; (9) NaCl-saturated solution; (10) measuring cylinder for biogas; (11) 1 M HCI solution; (12) pump; (13) magnetic stirrer; (14) heater; (15) water bath (37°C).

Microorganism Granula ted sludge (mesophilic) from the anaerobic digestion of starch wastewater, supplied by the Biotechnology Research Labora tory , Kobe Steel Ltd. (Kobe, Hyogo), was acclimatized using a lac- ta te-medium for a per iod of 6 months.

Media A sulfate-free minimal medium composed of inorganic salts, including trace elements, a vitamin mix- ture, and cysteine as the sole sulfur source, was used. The medium was the same as that used for Methanosarcina barkeri except for the addi t ion of sodium lactate or glucose instead of methanol (14).

Anaerobic reactor An anaerobic CSTR reactor (working volume: 1.6/) was mainta ined at 37°C, and the pH was control led at 7 .2±0.1 (Fig. 2). The circulation rate of the sample solution for measuring dissolved hydrogen was 0.01 l /min , which was same rate as that used for the measurement using distilled water.

Analyt ical procedures Gas product ion during cultivation was measured by collecting the evolved gas to a liquid displacement system (14). Hydrogen, methane and car- bon dioxide in the gas were determined by a gas chromatograph (GC-8AG, Shimadzu, Kyoto) with a thermal conduc- tivity detector (15). A gas sample of 1 ml was injected, and the detection limit was approximate ly 10 5 atm (corresponding to 1 Pa). Separat ion was accomplished at 90°C with activated charcoal 60/80 (3.00 m length × 3.0 mm i.d. staineless-steel column), using argon (2 kg /cm 2) as carrier gas. The detector temperature and current were 120°C and 60 mA, respectively.

Lactate and volatile fatty acids were determined by high performance liquid chromatography (TRIROTAR-V, Japan Spectroscopic Co., Tokyo) . A filtered sample of 40/al was injected. Separat ion was accomplished with Fine SIL NH2 (250mm length × 8 mm i.d. stainless-steel column). The elutant was 1% phosphate buffer (pH 1.5) at a flow rate of 1 ml /min . The detector was a UVIDEC-100- III (Japan Spectroscopic Co.) at 210 nm.

Glucose was analyzed by the glucose-oxidase method (Glucinet, Tournei , Belgium).

The MLVSS was calculated as the difference between the weights of mixed-l iquor suspended solids (MLSS) and ash (16).

Gases and chemicals N2 and H2 of >99 .99% (v/v) puri ty (Chugoku Teisan, Hiroshima) were used without

420 KURODA ET AL.

any t reatment . All chemicals used were guaranteed rea- gents, obta ined from commercial sources.

RESULTS A N D DISCUSSION

Measurement of dissolved hydrogen in distilled water and medium without cells

Calibration Figure 3 shows the result of the measurement of dissolved hydrogen in distilled water. The same results were obta ined for three measurements and those measurements provided high reliabili ty (two measurements shown). When H2 gas was changed to N2, it took about 1-1.5 h to return to the original level. As described above, since the calculated value of saturated dissolved hydrogen is 680pM, it was considered that the reading of the full scale on the DH2 detec tor(630pM) corresponded to the calculated value (680/zM).

Effects of interference Table 1 shows the dissolved hydrogen concentrat ions in distilled water and the medium used for this anaerobic digestion. The saturated concentration of dissolved hydrogen in the medium was slightly lower than that of distilled water. This result might be due to the presence of various inorganic salts in the medium.

Table 2 shows the dissolved hydrogen concentrat ions in the solut ion used for anaerobic digestion containing various chemicals. These sample solutions were bubbled with N 2 gas, and the output signals were obtained. It was found that the output signal of the sensor was not affected by volatile fat ty acids. As shown in Table 2, for the sample solution containing sulfide, the value increased with increas- ing of sulfide concentrat ions; thus the DHz-sensor was affected by sulfide. The dissolved hydrogen sulfide diffused through the permeable membrane might react at the anode. The reading of the sample solut ion containing 1.56 mM Na2S corresponds to 0.26 p M dissolved hydrogen (Table 2), suggesting that it is possible to neglect the influence at low sulfide concentrat ions, but not at high concentrat ions. Park in et al. (17) repor ted that in an anaerobic digestion process feeding propionate , levels of hydrogen sulfide in the range of 1.1 to 2.3 mM resulted in the failure of the process. In fact, a gas bubble was observed in- side the covered membrane at 1.56 mM of Na2S. Since the internal electrolyte was an acidic solution (0.1 M KC1 and 0.1 M HC1), this bubble might have been hydrogen sulfide gas. Therefore, the saturated dissolved hydrogen concen- t rat ion was forcibly decreased in contrast with that of distilled water (Table 1). When the sulfide gas bubble was

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F1G. 3. The reliability of the DH2-sensor in distilled water (37°C) with successive injections of N2 (99.9999~) gas and H2 (99.999991;) gas. Arrows: . , H2 was sparged; .-, N2 was sparged.

J. FERMENT. BIOENG.,

TABLE 1. Effects of the constituents of the medium on the response of the DH2-sensor

Medium Gases Dissolved hydrogen, calibrated (aM)

Distilled water N: 0 H~ 680 (100%)

Medium (pH 7.0) N~ 0 U 2 665 (97.8%F H?' 650 (95.7%F

" With the addition of sulfide (NazS-9H20:1.56 mM) and volatile fatty acids (acetate: 16.7 raM, propionate: 13.5 raM).

Relative values compared with that of distilled waler.

present at the inner covered membrane, the output signal of the DH2-sensor became a little low. Hence, both the internal electrolyte and covered membrane were completely substi tuted, and then the measurement o f dissolved hydrogen as described above was performed using the medium without sulfide. In this case, both the readings of zero and of the saturated dissolved hydrogen concentrat ion showed the same values as the previous data using the medium without sulfide.

The problem of the gas bubble may be improved by changing the internal electrolyte to alkaline solution. In this case, Ag-AgCI must also be changed to other materi- als, since Ag-AgC1 is not stable under alkaline condit ions and silver sulfide is formed on the electrode surface.

Measurement in an anaerobic reactor Figure 4 shows the profiles of the anaerobic digestion of lactate. The DH2-sensor was checked in a distilled water system at intervals of 120-h. As a result, the 0 and 100% indications were stable for 565-h cult ivation (data not shown). Dur- ing the anaerobic culture, since the cysteine concentrat ion as a sulfide source was very low, 85.2 pM (pH 7.2), the interference of sulfide was ignored. During 565-h cultivation, lactate was repeatedly supplied at concentrat ions of 5 to 25 mM by moni tor ing the propionate concentrat ion in the broth, which is an impor tan t intermediate in the bio- methanat ion of lactate. Al though propionate and acetate produced from the degradat ion of lactate fluctuated throughout the cultivation, no dramat ic rise in dissolved hydrogen was observed, which remained at low concentrations ranging from 0.5-3 p M during the operat ion, while the H2 part ial pressure ranged from 2 to 7 Pa. Since the fermentat ion broth was circulated to the anaerobic reactor after passing through the flow cell equipped with the DH2-sensor within I rain, H2 consumpt ion by microorgan- isms during the measurement of dissolved hydrogen could be neglected.

In Fig. 5, the relation between the output signal of the DH2 sensor and the H2 part ia l pressure in the gas phase of the reactor shows a good correlat ion ( r=0.85) . However, dissolved hydrogen concentrat ions from the DH2-sensor

TABLE 2. Effects of the addition of chemicals to the medium on the response of the DH2-sensor

Addition Concentration Dissolved hydrogen, calibrated (mM) (aM)

Medium (control) 0 Acetate 16.7 0 Propionate 13.5 0 Na:S. 9H20 0.047 0.05

0.781 0.1 l 1.561 0.26

Vou 71, 1991 DISSOLVED H2 MEASUREMENT IN ANAEROBIC DIGESTION 421

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FIG. 4. Performancesduring anaerobicdigestion oflactate. Cultureconditions:cellconcentration, l.5±0.2g of MLVSS perliter;pH, 7.2+0.1;temperature, 37°C; mixing, 400rpm. Symbols: ©,lactate;A, propionate;V, acetate. Arrows: +,lactateaddition.

were about 60-fold higher than the theoretical values calculated with H2 partial pressure of the gas phase based on Henry's law. For that, Whitmore et al. (18) directly measured the dissolved hydrogen concentration of 1/~M in an anaerobic digestion process maintaining a steady state in which they used membrane inlet mass spectrometry.

For the overconcentration of H2 in the liquid phase with respect to the thermodynamic equilibrium, Pauss et al. (19) have recently analyzed this from the mass balance equ- ations in the gas and liquid phases in terms of the gaseous metabolites produced in biomethanation processes. They obtained the following equation at a steady state where the composition of the gas phase, dissolved metabolites and dissolved gas concentration were constant:

[gas]L _ Qv +- 1 (1) [gas] L* KHR TkLa

where [gas]L, concentration of dissolved H2 in the reactor, M; [gas]L*, concentration of dissolved H2 in the reactor at thermodynamic equilibrium, M; Qv, volumetric H2 production rate, h ~; KH, Henry's constant, M.Pa-~; kLa, volumetric mass transfer coefficient, h-~; R, ideal gas constant, 8,314 Pa .M ' .K-J ; T, temperature, K.

Thus, it can be understood that the high value for overconcentration factor, [gaS]e/[gas]L* must occur due to the very low values of KH and kLa for H2 in biomethanation processes. In fact, the directly measured values of dissolved H2 during the biomethanation processes were much higher (35 to 81-fold) than those calculated from the thermodynamic equilibrium (19). The resulting kLa values for H2 ranged from 0.03 to 0.4 h ~ in three biomethanation processes compared with kLa values for 02 usually encoun- tered in aerobic cultures of 100 to 500 h 1.

In this work, assuming that from 381 to 449-h (Fig. 4) a steady state was achieved, kLa was calculated from Eq.

1. A very low value of kLa for H2 (i.e., 0.052 h ') was obtained, suggesting a strong mass transfer limitation from the liquid to gas phase.

When the substrate was changed to glucose instead of lactate using the same concentration of cysteine, i.e., the 85.2/tM, of the previous experiment (Fig. 6), there were dramatic increases of dissolved hydrogen concentration along with H2 partial pressure and volatile fatty acids, while only slight methane production was observed within 10-h culture in accord with a sharp drop of pH. This result shows a typical example of the failure of anaerobic digestion, indicating that the accumulation of hydrogen inhibited the degradation of volatile fatty acids (16) and the accumulation of acetate and H2 inhibited the methanation from H2/CO2 and acetate (20), consequently causing an acidic environment (pH 5.2) incapable of permitting an-

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Correlation between the indications of the DHrsensor a n d H 2 partial pressure of the gas phase in the methanogenic reactor.

422 KURODA ET AL. J. FERMENT. BIOENG.,

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FIG. 6. Performances during anaerobic digestion of glucose. Culture conditions: cell concentration, 1.7 g of MLVSS per liter; temperature, 37°C; without pH control; mixing, 400 rpm. Symbols: [], total gas ( H2+CH4+CO2); ,~), H2; V, CH4; ±, CO2 in c, and O, glucose; A, acetate; v, propionate; , , n-butyrate in d.

aerobic digestion to proceed. During 10-h culture, the dissolved hydrogen concentra-

t ion of the DH2-sensor was higher than the theoretical values calculated with H 2 partial pressure, as well as the results from lactate, but the gap was not so high, i.e., 1.0- 8.3 times. In the range of 300 and 400/iM of dissolved hydrogen concentration, the gap between the indications of the DH2-sensor and the theoretical values was negligi- ble. Pauss et al. (19) suggested that the higher gas production rate might contribute to the increase of kLa and consequently lead to the decrease of the gap. In this case, the gas production rate, namely the hydrogen gas production rate, was remarkably high compared with the results from lactate. After 10-h cultivation, the dissolved hydrogen concentration decreased gradually, but H2 partial pressure did not go down. In contrast with the results up to 10-h, the theoretical values were higher than the indication of the DH2-sensor, i.e., 1.0-2.4 times. This suggests that hydrogen in the liquid phase was consumed by H2-consuming methanogen, but the hydrogen gas transfer from the gas to the liquid phase was rate limiting because of its poor solubility, with a low value of kLa (19). The solubility of H2 gas from the gas to the liquid phase on the growth of Methanococcus thermolithotrophicus on H2/CO 2 was influenced by the agitation speed in the fermentor; at a high agitation speed methane productivity was strikingly improved (21).

After the pH was adjusted to 6.90 at 36-h culture, the degradation of volatile fatty acids (VFAs) gradually proceeded (data not shown). The dissolved hydrogen concentration and hydrogen partial pressure also decreased. During the degradation of VFAs, the dissolved hydrogen

concentrat ion was 4 . 0 + 0 . 5 ~ M and U 2 partial pressure was 11±2 Pa, which theoretically corresponded to 80.3 +_ 14.6 nM of dissolved hydrogen concentration, i.e., an overconcentration factor [gas]L/[gas]L* of about 50, in agreement with the results of the anaerobic digestion of lactate (Fig. 5). Moreover, we observed an increase of methane gas content in the gas phase accompanied by the degradation of VFAs (data not shown).

In conclusion, the membrane-covered DH2-sensor used in this study was useful for measuring the dissolved hydrogen concentrat ion continuously over a long period (24 d) in an anaerobic digestion process. However, a problem remains with respect to the interference in the measurements from sulfide when this is contained in the medium in a high concentration. In the future, this sensor needs to be tested on an industrial scale.

REFERENCES

I. Boone, D . R . and Bryant, M . P . : Propionate-degrading bacte- rium, Syntrophobacter wolinii sp. nov. gen. nov., from methanogenic ecosystems. Appl. Environ. Microbiol., 40, 626-632 (1980).

2. Bryant, M.P.: Microbial methane production-theoretical aspects. J. Anita. Sci., 48, 193-201 (1979).

3. Dolfing, J.: Kinetics of methane fermentation by granular sludge at low substrate concentrations. Appl. Microbiol. Biotechnol., 22, 77-81 (1985).

4. Harper, S. R. and Pohland, F. G.: Recent developments in hydrogen management during anaerobic biological wastewater treat- ment. Biotechnol. Bioeng., 28, 585-602 (1986).

5. Kristjansson, J. K., Schonheit, P. , and Thauer, R. K.: Different K~ values for hydrogen of methanogenic bacteria and sulfate

VoL. 71, 1991 DISSOLVED H 2 MEASUREMENT IN ANAEROBIC DIGESTION 423

reducing bacteria: an explanation for the apparent inhibition of methanogenesis by sulfate. Arch. Microbiol., 131, 278-282 (1982).

6. Robinson, J. A., Strayer, R.F. , and Tiedje, J. M.: Method for measuring dissolved hydrogen in anaerobic ecosystems: appli- cation to the rumen. Appl. Environ. Microbiol., 41, 545-548 (1981).

7. Archer, D.B., Hilton, M.G., Adams, P., and Wiecko, H.: Hydrogen as a process control index in a pilot scale anaerobic digestor. Biotechnol. Lett., 8, 197-202 (1986).

8. Scott, R. I., Williams, T. N., Whitmore, T. N., and Lloyd, D.: Direct measurement of methanogenesis in anaerobic digesters by membrane inlet mass spectrometry. Eur. J. Appl. Microbiol. Biotechnol., 18, 236-241 (1983).

9. Whitmore, T. N., Lazzari, M., and Lloyd, D.: Comparative stu- dies of methanogenesis in thermophilic and mesophilic anaerobic digesters using membrane inlet mass spectrometry. Biotech- nol. Lett., 7, 283-288 (1985).

10. Whitmore, T. N. and Lloyd, D.: Mass spectrometric control of the thermophilic anaerobic digestion process based on levels of dissolved hydrogen. Biotechnol. Lett., 8, 203-208 (1986).

11. Pauss, A., Samson, R., Guiot, S., and Beauchemin, C.: Continu- ous measurement of dissolved H2 in an anaerobic reactor using a new hydrogen/air fuel cell detector. Biotechnol. Bioeng., 35, 492-501 (1990).

12. Niedrach, L. W. and Stoddard, W. H.: Continuous voltametric monitoring of hydrogen and oxygen in water. Anal. Chem., 54, 1651-1654 (1982).

13. Sweet, W.J., Houchins, J.P., Rosen, P.R., and Arp, D.J.: Polarographic measurement of H2 in aqueous solutions. Anal.

Biochem., 107, 337-340 (1980). 14. Mazumder, T.K., Nishio, N., Fukuzaki, S., and Nagai, S.:

Effect of sulfur-containing compounds on growth of Methanosar- cina barkeri in defined medium. Appl. Environ. Microbiol., 52, 617-622 (1986).

15. Nishio, N., Eguchi, S.Y., Kawashima, H., and Nagai, S.: Mutual conversion between H2 plus C O 2 and formate by a formate-utilizing methanogen. J. Ferment. Technol., 61, 557-561 (1983).

16. Fukuzaki, S., Nishio, N., Shobayashi, M., and Nagai, S.: Inhibi- tion of the fermentation of propionate to methane by hydrogen, acetate, and propionate. Appl. Environ. Microbiol., 56, 719-723 (1990).

17. Parkin, G. F., Sneve, M. A., and Loos, H.: Anaerobic filter treat- ment of sulfate-containing wastewaters. Wat. Sci. Tech., 23, 1283-1291 (1990).

18. Whitmore, T.N., Lloyd, D., Jones, G., and Williams, T.N.: Hydrogen-dependent control of the continuous anaerobic digestion process. Appl. Microbiol. Biotechnol., 26, 383-388 (1987).

19. Pauss, A., Andre, G., Pettier, M., and Guiot, S. R.: Liquid-to- gas mass transfer in anaerobic processes: inevitable transfer limi- tations of methane and hydrogen in the biomethanation process. Appl. Environ. Microbiol., 56, 1636-1644 (1990).

20. Fukuzaki, S., Nishio, N., and Nagai, S.: Kinetics of methanogenic fermentation of acetate. Appl. Environ. Microbiol., 56, 3158-3163 (1990).

21. Fardeau, M. and Belaich, J.: Energetics of the growth of Methanococcus thermolithotrophicus. Arch. Microbiol., 144, 381-385 (1986).

measurement of dissolved hydrogen in an anaerobic digestion process by a membrane-covered electrode

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