A new method for the determination of dissolved ... - Springer Link

Bioprocess Engineering 15 (1996) 317—322 ( Springer-Verlag 1996

A new method for the determination of dissolved sulfide in strongly colored anaerobically treated effluents G. Percheron, N. Bernet, R. Moletta

317 Abstract A simple method for dissolved sulfide determination in colored complex media was developed using ion exchange chromatography. Its principle is based on the complete oxidation of an unstable compound (sulfide) into its stable form (sulfate) through a strong oxidant : hydrogen peroxide. The difference between sample analyzed before and after this treatment gives the total dissolved sulfide. In order to avoid H S exhaust, this oxidation has to be performed immediately 2 after sampling, without cell separation. In that way, standard solutions were prepared using raw anaerobic effluents from an industrial plant. It was shown in the calibration curve that no bacterial interaction was present. Finally, sulfide from continuous and discontinuous digestions of these sulfate rich wastewaters were successfully assayed by this technique. A theoretical evaluation based on Henry’s law and the sulfide dissociation equilibrium led to a very good agreement with the analytical results. List of symbols [SO 2~] in (mmol/l) 4 [SO 2~] out (mmol/l) 4 [S ] (mmol/l) d [H S] (mmol/l) 2 s [H S] (mmol/l) 2 G % [H S] ([) 2 G f ([) a ([) K Q L Q G TKN TOC

Sulfate effluents concentration Outgoing sulfate concentration Total dissolved sulfide Soluble free sulfide Gaseous sulfide Fraction of H S in the gas phase 2 Fraction of free H S in solution 2 Absorption coefficient, a is 1.74 at 37.5 °C (mol) Sulfide equilibrium constant, K is 1.59 10~7 M at 37.5 °C (ml/l.d) Liquid flow (ml/l.d) Gas flow (mg/l) Total Kjeldahl Nitrogen (mg/l) Total Organic Carbon

Received: 27, February 1996 G. Percheron, N. Bernet, R. Moletta Laboratoire de Biotechnologie de l’Environement, Institut National de la Recherche Agronomique Avenue des Etangs, 11100 Narbonne, France. Correspondence to: N. Bernet This study was supported by a research grant from the ‘‘Agence de l‘Environement et de la Maitrise de l‘Energie’’, (ADEME) Paris, France. The authors would like to express their gratitude to A. BORIES for his valuable advice.

TCOD SCOD TVFA TSS VSS Vm

(mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (1/mol)

Total Chemical Oxygen Demand Soluble Chemical Oxygen Demand Total Volatile Fatty Acids Total Suspended Solids Volatile Suspended Solids Volume of one mole of perfect gas: Vm\25.5 l/mol at 37.5 °C

1 Introduction Anaerobic biological treatment of high sulfate content effluents has been intensively studied [1— 4]. Sulfate reduction to sulfide by sulfate reducing bacteria (SRB) causes unfavorable digester conditions and decreases methane production. Several explanations have been proposed for this phenomenon but the exact mechanism is still unknown. Sulfate reduction may reduce the quantity of organic materials available for their conversion to methane. There would be a competition between methane producing bacteria (MPB) and SRB [5]. Furthermore, un-ionized hydrogen sulfide (H S) has toxic effects on various 2 anaerobic bacteria since it readily permeates membranes and denatures native proteins inside the cytoplasm [6]. Nevertheless, it is also an essential nutrient for methanogens [1]. Moreover, sulfide is known to precipitate essential metals for MPB such as iron, zinc, cobalt and nickel. Finally, it seems that COD/S ratio of the effluents has a great influence on relationships between MPB and SRB. In that way, a high COD/S ratio promotes methanogenesis [7—11]. In order to better understand these interactions together with the sulfate reduction both sulfate and sulfide have to be assayed. Newly developed low capacity columns used in ion chromatography allow quick and reproducible determination of SO2~ [12] and they are usually used in most of the studies 4 about sulfate reduction. Sulfate can also be evaluated by turbidimetric techniques. Sulfide can be easily separated from common anions by ion exchange techniques. However, their detection using classical methods such as conductivity is very poor due to their low dissociation following protonation by the suppressor column. Therefore, sulfide is often assayed by iodometric titration, spectrophotometry [13—15] or ion selective electrodes [16]. Unfortunately, in case of complex effluents such as wastewaters from food processing industries, some of these methods can give erratic results. As an example, the Cord-Ruwisch technique which produces a dark brownish precipitation of CuS [14] cannot be used with very strongly colored effluents. Other methods such as the methylene blue method [15] need sulfide precipitation with zinc ion in order

Bioprocess Engineering 15 (1996)

Table 1. Studies using conventional methods for sulfide assay

318

Reference

Technique

Medium

Dalsgaard et al. (1992) [19] Cypionka et al. (1986) [20] Polprasert et al. (1995) [21] Lovley et al. (1982) [17] Cord-Ruwisch et al. (1988) [22] Winfrey et al. (1977) [23] Yoda et al. (1987) [24] Qatibi et al. (1990) [25] Harada et al. (1994) [26] Ronnow et al. (1981) [27]

Microelectrode Sulfide electrode Ion selective electrode 33SO2~ 4 Cord-Ruwisch’s method Spectrophotometry Colorimetric method Cord-Ruwisch’s method Iodometric method Methylene blue method

Synthetic medium Mineral medium Synthetic medium Sediments Synthetic medium Sediments pore water Synthetic medium Synthetic medium Synthetic medium Methanogenic basal medium

Truper’s method

Synthetic medium

Cline’s method

Synthetic medium

Mizuno et al. (1994) [11] Mounfort et al. (1979) [28] Cappenberg (1975) [29] Ueki et al. (1988) [30] Klemps et al. (1985) [31] Dalsgaard et al. (1994) [32]

to remove it from the culture medium. They take approximately half an hour and allow analysis of traces of dissolved sulfides down to concentrations of micromoles per liter. Consequently, many of the studies using these techniques were done with sediments or pure SRB cultures, very often with synthetic media (Table 1). In complement, it is to be noticed that using 35SO2~ during 4 sulfate reduction analysis, the produced 35S2~ can be distilled, trapped and quantified by liquid scintillation counting [17]. Moreover, placing an ion exchange column in front of an electrochemical detector, (e.g., a silver working electrode), sulfide can also be monitored [18]. This study presents a new and convenient method for the determination of sulfide in colored effluents, oxidizing the sulfide with hydrogen peroxide (H O ) immediately after sampling and directly in the broth 2 2 without cell separation.

2 Materials and methods 2.1 Procedure Sulfate assay: Just after sampling, the produced sulfide was precipitated down to the vessel bottom as FeS. This is achieved by addition of 10 ll of FeCl 4M (Prolabo) to 200 ll of culture 2 medium in order to avoid an interference by air sulfide oxidation. After centrifugation (1 min., 5500 g), the preparation was assayed by ion chromatography (Dionex DX100). Total dissolved sulfur assay: The pH of freshly sampled effluents has been increased to pH 10 by NaOH (6N) addition (20 ll NaOH, 400 ll sample) to reduce loss of volatile H S that 2 escapes into the atmosphere. The preparation was then oxidized by 100 ll of H O (30% v/v). After centrifugation 2 2 (10 min. 8000 g) or filtration, the produced sulfate was analyzed by ion chromatography. Difference between sulfate and dissolved sulfur assay as sulfate gives the total dissolved sulfide. Two or three hours are needed to oxidize completely very strongly reduced effluents but only few minutes are required for synthetic media.

Moreover, non centrifuged oxidized samples are very stable at 4 °C (several days) or they can be cooled at [20 °C for long time.

2.2 Sulfide standard preparation A washed crystal of Na S 9H O was dissolved to a known 2 2 concentration (100 mM) in distilled water or in centrifuged anaerobic digested effluents. From this solution, a dilution series of soluble sulfide was prepared (0—50 mM). Since solutions were alkaline through NaOH supply, volatile H S 2 exhaust in the gas atmosphere is negligible.

2.3 Anaerobic digestion conditions and apparatus Two experiments have been set up in order to validate the method: a batch culture in one liter reactor and an anaerobic continuous digestion of colored sulfate rich industrial effluents whose composition was reported in Table 2. Batch culture: One liter of sludge from a local anaerobic digester was added to raw concentrated effluents in order to obtain a soluble COD of 15000 mg/l and a TOC concentration of 6000 mg/l. pH was lowered to 7.3 by HCl addition. The reactor was completely deoxygenated through argon bubbling

Table 2. Composition of the industrial effluents used during this study Parameter

Raw concentrated effluents (mg/l)

Diluted effluents (mg/l)

TOC TCOD SCOD SO2~ 4 TVFA TSS VSS TKN

260000 450000 430000 62000 18000 45000 36000 34000

11000 20000 19000 2800 800 2000 1600 1500

G. Percheron et al.: A new method for the determination of dissolved sulfide

and crimped. The growth temperature was kept constant at 35 °C with a thermostated envelope and the stirrer speed was held at 400 rpm. Sulfate and sulfide were frequently analyzed and the final sulfur balance was established. Chemostat culture: Two liters of anaerobic sludge were cultivated into two continuously stirred tank reactors (CSTR) fed with diluted effluents (Table 2) in order to be as close to the operating conditions of the local industrial plant as possible. A very low organic loading rate was applied to avoid a sulfide inhibition (500 mg COD/l.d).

319

2.4 Analyses TOC was determined through UV oxidation using a Dohrman DC-80 and the COD measurements were done by the potassium dichromate ferrous ammonium sulfate method. Gas composition (CO , CH , O , H , and N ) was evaluated 2 4 2 2 2 by gas chromatography (Shimadzu CR3A) using an Hayesep 80—100 mesh column, a molecular sieve column and a katharometer detector. Gaseous H S was estimated using 2 Drager Tubes. The gas flow was measured with a device based on liquid displacement by the outgoing gases and described by Moletta et al. [33]. Sulfate was analyzed by an ion chromatography system using conductivity detection (Dionex DX100). Separation and elution were carried out on an anionic IonPac AS4A analytical column using a carbonate/bicarbonate eluant and a sulfuric regenerant.

Fig. 1. Sulfide calibration curve determined with water dissolved Na S 2 crystals. pH of standard solution was up to 10 and one volume of H O was 2 2 added to four volumes of sample

3 Results and discussions 3.1 Standardization with water dissolved Na2S crystals In order to check that no H S lost via gas production occurred 2 during the reaction and that the amount of H O was high 2 2 enough to perform a complete oxidation (1 volume of H O , 2 2 4 volumes of sample), a calibration curve was made using pure solutions of Na S 9H O (Fig. 1). This curve was linear in the 2 2 studied range (0 to 50 mM) and its slope value was close to 1. It is to be noticed that sulfide concentrations in our anaerobic digesters did not exceed 30 mM.

3.2 Sulfide calibration with Na2S crystals dissolved in raw anaerobic digested effluents We already pointed out that it was of major importance to avoid H S exhaust during sampling. Therefore, experiments 2 between sampling and oxidation had to be limited. Thus, H O 2 2 was added directly to the sample without a centrifugation step. In that way, the standard solution was prepared dissolving Na S 9H O crystals in centrifuged anaerobic digested effluents 2 2 from an industrial plant. Each standard dilution was then done mixing a known volume of this solution with a known volume of raw digested wastewaters (non centrifuged effluents) so that they contained different amounts of VSS. A set of corresponding reference solutions without sulfide crystal was also prepared with the same ratios of centrifuged and non

Fig. 2. Sulfide calibration curve determined with Na S crystal dissolved in 2 raw anaerobic effluents. Values were expressed against a series of oxidized ‘‘blanks’’ whose sulfate concentration after H O treatment was 8.4 mM 2 2 (residual sulfide of the raw anaerobic used effluents)

centrifuged effluents. Thus, each dilution and its associated ‘‘blank’’ had the same VSS content. Sulfate in all samples was assayed both before and after oxidation. Although reference preparations did not have the same VSS content, they gave the same results after H O treatment. This clearly showed that VSS 2 2 did not interfere with our technique. It meant that there was neither transformation of bacterial sulfur nor solid sulfur compounds oxidation S°, FeS. . .). Furthermore, hydrogen peroxide treatment done directly after sampling without bacteria separation gave a linear and reproducible calibration curve with a slope close to 1 (Fig. 2).

Bioprocess Engineering 15 (1996)

3.3 Theoretical evaluation of dissolved and gaseous sulfide Soluble sulfide forms a weak acid ionized in aqueous solution, the extent depending upon the pH. Its dissociation is distributed between neutral hydrogen sulfide (H S) and the 2 double-negative charge of free sulfide (S2~):

H S % H`]HS~ % S2~]2H` 2 ———————————————— Acid pH Alkaline pH 320

(1)

At the pH required for anaerobic digestion (7—8), only the first dissociation of H S is of importance and the mass action 2 constant for this equilibrium is:

[H`] [HS~ ] \K, [H S] 2 s

(2)

The free H S fraction in solution can be related to this constant 2 and the reactor mixed liquor pH by:

A

B

K ~1 . f \ 1] 10~1H

(3)

Moreover, some sulfide escapes from the digester with the produced biogas. The equilibrium between the gas and the soluble (sulfide) phases is governed by Henry’s law:

[H S] \a[H S] . 2 s 2 G

(4)

Values for the mass action constant K and the absorption coefficient a depend upon the temperature and have been taken from Lawrence et al., Wilhelm et al. and Tursman et al. [34, 35, 6]. At 37.5 °C, a is equal to 1.74 and K to 1.59 · 10~7 M. From Eqs. (3) and (4), it is possible to calculate a theoretical [H S] as follows: 2 G

f [S ] [H S] \ d and % [H S] \[H S] . Vm. 2 G 2 G s G a

(5)

Gaseous sulfide concentrations evaluated from Eq. (5) and measured using Drager Tubes were compared during discontinuous anaerobic cultures. Equations (3), (4), (5) and the elemental sulfur balance allowed the calculation of the theoretical dissolved sulfide during continuous anaerobic digestion:

[S ]\Q d L

[SO 2~ ] in[[SO 2~ ] out 4 4 . Q f G ]Q L a

(6)

3.4 Sulfur balance on discontinuous anaerobic cultures With the aim of studying sulfate reduction and methanogenesis of high sulfate content wastewaters, several discontinuous digestions have been carried out. Sulfate, total dissolved and gaseous sulfide were assayed using our method and Drager Tubes (Fig. 3). The theoretical gaseous H S 2 evaluated from Eq. (5) and the gaseous sulfide concentration measured with Drager Tubes are in very good agreement since both curves were identical. Furthermore, the sulfur balance was closed (Table 3). Finally, sulfide oxidation did not occur when sulfate was quickly assayed (immediately after sampling) or cooled at [20 °C. Iron seemed to only have a little effect

Fig. 3. Sulfate, dissolved sulfide and gaseous sulfide evolutions during discontinuous anaerobic digestion of colored industrial wastewaters comparison of experimental H S gas measured with Drager tubes and 2 theoretical values calculated from Eq. 5

Table 3. Sulfur balance established during a batch anaerobic culture. Sulfate and sulfide removed from the reactor through sampling have been considered

SO2~ 4 Sampled SO2~ 4 Soluble sulfide Sampled sulfide Gas sulfide exhaust Total

Initial state (T\0 h) (mmol)

Final state (T\400 h) (mmol)

20.5 0 0 0 0 20.5

0 1.4 12.3 2.2 4.8 20.7

(Fig. 3) and samples sulfate concentration reached zero at the end of the culture without iron protection. Our medium was very reduced and probably not readily oxidable by air contact. Nevertheless, the iron treatment could be suitable for other effluents. Moreover, iron treated samples turned black, viscous and after centrifugation and oxidation by H O , it did not show 2 2 any sulfate increase which means that all sulfide has been precipitated (results not shown).

3.5 Sulfide assay during continuous anaerobic digestion of sulfate rich effluents Two continuously stirred tank digesters were fed with an organic loading rate (OLR) of 500 mg COD/l.d. This low OLR allowed a stable running of the process for at least 2 months without sulfide inhibition. The operating conditions are reported in Table 4, the results of sulfide assays on both reactors and the corresponding theoretical values being evaluated from equations (5) and (6). Small difference between the experimental results and the concentrations calculated

G. Percheron et al.: A new method for the determination of dissolved sulfide

Table 4. Continuous anaerobic digestions : operating conditions, comparison between sulfur theoretical and experimental values

Temperature (°C) pH Q (ml/l.d) L Q (ml/l.d) G [SO 2~] in (mmol/l) 4 [SO 2~] out (mmol/l) 4 [S ] calculated (mmol/l) D [S ] assayed (mmol/l) D % H S measured 2 G % H S calculated 2 G

Reactor 1

Reactor 2

37.5 7.8 26.5 220 23.9 0.2 16.5 17 2.2 2.3

37.7 7.7 24 220 23.9 0.4 15.2 14.3 1.9 2.2

from the sulfate consumption was noticed. We already suggested that insoluble sulfur forms were not oxidized during hydrogen peroxide treatment. Moreover, it seems that other dissolved sulfur forms such as sulfite (SO2~ ) and thiosulfate 3 (S O2~) (i.e., compounds involved in mechanisms of dissimila2 3 tory reduction of sulfate) were not significantly present during our experiments. It was possible to detect these forms using ion chromatography. Sulfite and thiosulfate were eluted respectively before and after sulfate. Nevertheless, these compounds were not observed during sulfate assay.

4 Conclusion In this study, a new and simple method for sulfide assay has been used in order to better understand the sulfate reduction. Sulfur balance and theoretical evaluations showed that this oxidative technique was suitable for colored effluents. Of course, sulfite and thiosulfate can interfere with this sulfide assay and their presence has to be verified prior to the oxidation. These compounds can be assayed by ion chromatography and a method based on our study can be carried out if needed. Finally, non centrifuged oxidized and centrifuged non treated samples can be cooled at [20 °C and assayed later without any problem which makes our technique very convenient.

References 1. Speece, R.E.: Anaerobic biotechnology for industrial wastewater treatment, Environ. Sci. Technol. 17, 9 (1983) 416A—427A 2. Hilton, M.G.; Archer, D.B.: Anaerobic digestion of sulfate-rich molasses wastewater: inhibition of hydrogen sulfide production. Biotech. Bioeng. 31 (1988) 885—888 3. Yoda, M.; Imabayashi, S.; Suzuki, N.: Pilot and full-scale experience in anaerobic treatment of brewer’s yeast processing wastewater. Wat. Sci. Tech. 23 (1991) 1167—1177 4. Reis, M.A.M.; Lemos, P.C.; Carrondo, M.J.T.: Biological sulfate removal of industrial effluents using the anaerobic digestion. In Forum for Applied Biotechnology, Gent, Belgium, (1995) 2701—2707 5. Shonheit, P.; Kristjansson, J.K.; Thauer, R.K.: Kinetic mechanism for the ability of sulfate reducers to out-compete methanogens for acetate Arch. Microbiol. 132 (1982) 285—288 6. Tursman, J.F.; Cork, D.J.: Biological waste treatment. In Mizrahi, A. (eds) Advances in Biotechnological processes. 12, 1989. Liss, A.R., New York, pp 273—285

7. Isa, Z.; Grusenmeyer, S.; Verstraete, W.: Sulfate reduction relative to methane production in high-rate anaerobic digestion: Technicals aspects. Appl. Environ. Microbiol. 51, 3 (1986) 572—579 8. Isa, Z.; Grusenmeyer, S.; Verstraete, W.: Sulfate reduction relative to methane production in high-rate anaerobic digestion: Microbiologicals aspects. Appl. Environ. Microbiol. 51, 3 (1986) 580—587 9. Parkin, G.F.; Lynch, N.A.; Kuo, W.C.; Van Keuren, E.L.; Bhattacharya, S.K.: Interaction between sulfate reducers and methanogens fed acetate and propionate. J. Wat. Pollut. Control Fed 62, 6 (1990) 780—788 10. Vavilin, V.A.; Vasiliev, V.B.; Rytov, S.V.; Ponomarev, A.V.: Selfoscillating coexistence of methanogens and sulfate-reducers under hydrogen sulfide inhibition and the pH-regulating effect. Biores. Technol. 49 (1994) 105—119 11. Mizuno, O.; Li, Y.Y.; Noike, T.: Effects of sulfate concentration and sludge retention time on the interaction between methane production and sulfate reduction for butyrate. Wat. Sci. Tech. 30, 8 (1994) 45—54 12. Bak, F.; Scheff, G.; Jansen, K.H.: A rapid and sensitive ion chromatographic technique for the determination of sulfate and sulfate reduction rates in freshwater lake sediments. FEMS Microbiol. Ecology. 85, (1991) 23—30 13. Cline, J.D.: Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol. Oceanogr. 14 (1969) 454—458 14. Cord-Ruwisch, R.: A quick method for the determination of dissolved and precipitated sulfides in cultures of sulfate-reducing bacteria. J. Microbiol. Methods. 4 (1985) 33—36 15. Truper, H.G.; Schlegel, H.G.: Sulphur metabolism in Thiorhodaceae I. Quantitative measurements on growing cells of Chromatium okenii. Antonie van Leeuwenhoek. 30 (1964) 225—238 16. Cypionka, H.: Sulfide-controlled continuous culture of sulfatereducing bacteria. J. Microbiol. Methods. 5 (1986) 1—9 17. Lovley, D.R.; Dwyer, D.F.; Klug, M.J.: Kinetic analysis of competition between sulfate reducers and methanogens for hydrogen in sediments. Appl. Environ. Microbiol. 43, 6 (1982) 1373—1379 18. Rocklin, R.D.; Johnson, E.L.: Determination of cyanide, sulfide, iodide, and bromide by ion chromatography with electrochemical detection. Anal. Chem. 55 (1983) 4—7 19. Dalsgaard, T.; Bak, F.: Effect of acetylene on nitrous oxide reduction and sulfide oxidation in batch and gradient cultures of Thiobacillus denitrificans. Appl. Environ. Microbiol. 58, 2 (1992) 1601—1608 20. Cypionka, H.; Pfennig, N.: Growth yields of Desulfotomaculum oriental with hydrogen in chemostat culture. Arch. Microbiol. 143 (1986) 396—399 21. Polprasert, C.; Haas, C.N.: Effect of sulfate on anaerobic processes fed with dual substrates, Wat. Sci. Tech. 31, 9 (1995) 101—107 22. Cord-Ruwisch, R.; Seitz, H.J.; Conrad, R.: The capacity of hydrogenotrophic anaerobic bacteria to compete for traces of hydrogen depends on the redox potential of the terminal electron acceptor. Arch. Microbiol. 149 (1988) 350—357 23. Winfrey, M.R.; Zeikus, J.G.: Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments. Appl. Environ. Microbiol. 33, 2 (1977) 275—381 24. Yoda, M.; Kitagawa, M.; Miyaji, Y.: Long term competition between sulfate-reducing and methane-producing bacteria for acetate in anaerobic biofilm. Wat. Res. 21, 12 (1987) 1547—1556 25. Qatibi, A.L.; Bories, A.; Garcia, J.L.: Effects of sulfate on lactate and C2, C3 volatile fatty acid anaerobic degradation by a mixed microbial culture. Antonie van Leeuwenhoek. 58, (1990) 241—248 26. Harada, H.; Uemura, S.; Momonoi, K.: Interaction between sulfatereducing bacteria and methane-producing bacteria in UASB reactors fed with low strength wastes containing different levels of sulfate. Wat. Res. 28, 2 (1994) 355—367 27. Ronnow, P.H.; Gunnarsson, L.A.H.: Sulfide-dependent methane production and growth of a thermophilic methanogenic bacterium. Appl. Environ. Microbiol. 42, 4 (1981) 580—584 28. Mounfort, D.O.; Asher, R.A.: Effect of inorganic sulfide on the growth and metabolism of Methanosarcina barkeri strain DM. Appl. Environ. Microbiol. 37, 4 (1979) 670—675

321

Bioprocess Engineering 15 (1996)

29. Cappenberg, T.E.: A study of mixed cultures of sulfate-reducing and methane-producing bacteria, Microbial Ecol. 2 (1975) 60—72 30. Ueki, K.; Ueki, A.; Simogoh, Y.: Terminal steps in the anaerobic digestion of municipal sewage sludge: Effects of inhibitors of methanogenesis and sulfate reduction. J. Gen. Appl. Microbiol. 34 (1988) 425—432 31. Klemps, R.; Cypionka, H.; Widdel, F.; Pfennig, N.: Growth with hydrogen, and further physiological characteristics of Desulfotomaculum species. Arch. Microbiol. 143 (1985) 203—208

32. Dalsgaard, T.; Bak, F.: Nitrate reduction in a sulfate-reducing bacterium, Desulfovibrio desulfuricans, isolated from rice paddy soil: sulfide inhibition, kinetics, and regulation. Appl. Environ. Microbiol. 60, 1 (1994) 291—297 33. Moletta, R.; Albagnac, G.: A gas meter for low rates of gas flow: Application to the methane production. Biotechnol. letters. 4, 5 (1982) 319—322 34. Lawrence, A.W.; McCarty, P.L.: The effects of sulfides on anaerobic treatment. Air and Water Pollut Int J. 10 (1966) 207—221 35. Wilhelm, E.; Battino, R.; Wilcock, R.: Low-pressure solubility of gases in liquid water. Chem Rev. 77, 2 (1977) 219—242

322

.