System. Appl. Microbiol. 11, 202-206 (1989)

Sulfur Reduction and Inhibition in Anaerobic Treatment of Simulated Pulp Mill Wastewater

JAAKKO A. PUHAKKAl, JOHN F. FERGUSON2, MARK M. BENJAMIN2

,

and MIRJA SALKINOJA-SALONEN3

1 Department of Civil Engineering, Water and Environmental Engineering, Tampere University of Technology, SF-33101 Tampere, Finland

2 Department of Civil Engineering, University of Washington, Seattle, WA 98195, USA 3 Department of General Microbiology, University of Helsinki, SF-00280 Helsinki, Finland

Received August 20, 1988

Summary

Bioassay studies were conducted on the anaerobic degradation of a simulated pulping wastewater (evaporator condensate) containing sulfur compouds (sulfate, sulfite, or dithionite). Sulfite and dithionite (1000 mg SIl) totally inhibited methane production while sulfate (1000 mg SIl) had a minor effect. High concentrations of sulfite (1000 mg SIl) appeared to inhibit sulfite reduction. Biochemical reduction of dithionite (100-1000 mg SIl) could not be demonstrated. Methane bacteria outcompeted sulfate-reducing bacteria when methanol was the only substrate available. Methanol was utilized by sulfate reducers in the presence of a specific methanogen inhibitor, BES. Increase of the methanol concentration in an acetate -methanormedium stimulated sulfate reduction.

Key words: Pulping wastewaters - Anaerobic treatment - Methane fermentation - Sulfate reduction -Sulfite reduction - Dithionite reduction - Sulfur inhibition - Competition for substrates

Introduction

High rate anaerobic treatment of industrial wastewaters rich in dissolved organic compounds has found many new applications in recent years. Several full scale anaerobic reactors are already in use in the forest industry (Rantala and Luonsi, 1985). With the adoption of stricter discharge regulations for pulp mills the existing waste treatment sys­tems may become undersized. The industry generates a variety of waste streams, some containing high concentra­tions of organic acids and alcohols that are readily used by methanogenic cultures. These concentrated waste streams are potential candidates for anaerobic pretreatment.

Inorganic sulfur can appear as sulfate, sulfite, or dithionite in pulping wastewaters. Sodium sulfate (kraft process) and sulfur dioxide (sulfite process) are principal agents added in the chemical pulping. Dithionite is used to bleach mechanical pulp. The evaporator condensate from the acid sulfite and bisulfite pulping processes are treatable

by anaerobic means (Brune et al., 1982; Eis et al., 1983; Frostell, 1984). These studies indicate that sulfur com­pounds reduce treatment efficiency and methane yield.

The generation of hydrogen sulfite (H2S) during anaerobic treatment of industrial wastewater is undesir­able not only because of odors and toxicity but also of economic losses (e. g. corrosion of metal and concrete by H2S formation). In addition, dissolved sulfide can be toxic to anaerobic microorganisms at concentrations above 200 mgll (McCarty, 1964). Other sulfur compounds have also been reported to inhibit anaerobic processes (Khan and Trottier, 1978; Puhakka et al., 1985). The competition for available substrate (e. g. Hz, acetate) between sulfate­reducing bacteria (SRB) and methane bacteria (MB) has been studied extensively in natural environments (Abram and Nedwell, 1978; Lovley et al., 1982; Oremland et al., 1982).

The purpose of this study was to investigate the factors controlling methane production and sulfur reduction in anaerobic treatment of forest industry wastewaters.

Materials arid Methods

Seed. Inoculum A was from a pilot anaerobic reactor treating neutral spent sulfite process effluent (Wastewater Technology Centre, Burlington, Ontario, Canada). Inoculum B was a mixture of sludges from a municipal digester (West Point Treatment Plant, Seattle, W A) and the bottom of an aerated lagoon treating sulfite pulp mill effluent (Georgia Pacific Mill, Bellingham, WA).

Media. A basic medium containing resazurin, sodium sulfide, nutrients and vitamins was used in all batch bottle bioassays (Owen et aI., 1979). The synthetic wastewater simulating the organic composition of sulfite evaporator condensate from the acid bisulfite pulping process contained per litre 3000 mg of acetic acid, 1000 mg of methanol, and 500 mg of glucose. Na2S04, Na2S03, or NaZS204 was added 1000 mg/l as S. The total liquid volume was 50 mUassay bottle. The medium for methanol utilization test (50 mUBottle) contained methanol (2000 mg/ml) and Na2S04 (800 mg SIl). Methane and hydrogen sulfide production from acetate - methanol mixtures of 30: 1 and 3 : 1 (as COD) were tested using acetate (2800 mg/l) and methanol (70 and 670 mg/l) in the presence of NaZS04 (1000 mg SIl). The total liquid volume in these tests was 25 ml/assay bottle. The acetate (1900 mg/l) medium (25 mUbottle) was used for testing the reduction of Na2S04, Na2S03, and NaZS204 by SRB at concentrations of 100-1000 mg S/I. A specific inhibitor of methane bacteria (2-bromoethane sulfonic acid, BES) was added (10 mol) to the acetate medium. The modified Postgate medium was used for the enumeration of lactate utilizing SRB (Lapage et aI., 1970). The medium by Badziong et al. (1978) was used for enumeration of hydrogen plus acetate plus CO2 utilizing SRB. Viable counts of MB were determined in the media by Mdh and Smith (1981) and by Ferry et aI., (1974).

Batch bottle bioassays. Tests were performed as decribed by Owen et al. (1979) and modified by Benjamin et al. (1984). Stock solutions of the substrates, sulfur compounds, and BES were pre­pared from Nz-flushed, boiled deionized water. Bottle injections were performed with syringes and needles prerinsed with a reduc­ing solution (10 g cystein HClIl boiled deionized water).

Viable counts. The roll tube method (Hungate, 1969) was used for viable counts with 5 ml of medium in 16 ml anaerobic culture tubes (Belleo). The bioassay bottles were agitated for 2 minutes by a whirl mixer of obtain homogenous suspensions prior to sampling. Serial dilutions were made using an anaerobically pre­pared diluent with 2 g of gelatin, 0.001 g of resazurin in 500 ml deionized water mixed with 500 ml of sodium phosphate buffer (0.06 M, pH 7.0). 0.1 rnl of each dilution was inoculated with prerinsed syringes and needles to liquified agar media at 45°C. The number of black colonies indicated the viable SRB. In the enumeration of acetate utilizing MB hard colonies with caleium carbonate precipitates were counted from the acetate media.

Analytical procedures. A 1 ml sample was acidified with for­mic acid and analyzed for acetate by gas chromatography using a packed glass column (1.8 m by 2 mm) with 0.3% Carbo wax 20 MlO.1% H3P04 on 60/80 Carbopack C (Supeleo, Inc.) in a Hew­lett-Packard 5894A gas chromatograph equipped with a flame ionization detector, oven temperature of 120°C, and that of the detector and injection port 200°C. Carrier gas was nitrogen (flow rate 50 mUmin). Methane was analyzed by gas chromatography. Sulfide was measured potentiometrically with an Orion Research Inc. lead electrode (Model 948200) and a double junction refer­ence electrode (Model 900200) attached to a mV meter (Corning

Anaerobic Treatment of Sulfur Rich Wastewater 203

Ltd., Model 150). Sulfate concentrations were measured from filtered samples using an automated methylthymol blue method (EPA 1979; Method 375.2; Orion, 1980).

Results

The effect of initial oxidation state of sulfur compounds on the production of methane from synthetic wastewater and on viable counts of MB and SRB was studied. The results in Fig. 1 show that sulfite and dithionite completely inhibited methane production at concentrations of 1000 mgll (as S), while sulfate at the same concentration only slightly decreased methane yield. Sulfide was produced both from sulfite (290 mg S=/l) and sulfate (55 mg S=/l) but not from dithionite. Viable counts of MB are shown in Fig. 2. A decrease in the number of acetate utilizing MB in the sulfite wastewater was seen at the end of the experi­ment (Fig. 2) but otherwise the sulfur ions had little effect. The reduction of acetoclastic MB could be due to sulfide accumulation during incubation. The numbers of viable SRB increased with sulfur compounds as compared with the control without added sulfur (Fig. 2).

Fig. 3 illustrates methanol utilization by MB and SRB in the presence and absence of BES. Sulfide production in methanol plus sulfate medium was observed only when MB were inhibited with BES. All the methanol was con­verted to methane in the absence of BES.

The effects of different acetate - methanol ratios in synthetic wastewater on MB - SRB competition are shown in Fig. 4. Sulfide production from sulfate was stimulated by increases in the concentration of methanol in the waste­water. The molar ratio of methane produced to substrate utilized (acetate or methanol) ranged from 0.7 to 0.9. The theoretical value for acetate splitting is 1.0 and 0.75 for

c o

13 100;-----------------------------~~ =:J

"'0 e a. 80 SULFUR (1) ADDITION c d--- 60 ..c.-_E (1)_

E (1)

.~

13 =:J E =:J

W

L.O

20

L. 8 12 16 20

TIME(Oays)

55 290

o

1..3

Fig. 1. Effect of different sulfur compounds (1000 mg S/I) on methane production from synthetic wastewater by inoculum A (0), synthetic wastewater without sulfur; (.), no carbon subs­trate added. Inset shows total sulfide concentrations after 43 days of incubation.

204 J. A. Puhakka, J. F. Ferguson, M. M. Benjamin, and M. Salkinoja-Salonen

methanol use. Methane production results in acetate plus methanol medium indicate that methanol apparently was the preferred electron acceptor. The increase in sulfide production in acetate plus methanol media compared to acetate medium could be due to increase in acetate utiliza­tion · by. SRB while methane was first produced from methanol instead of acetate.

Table 1 lists sulfide and acetate concentrations after sludge incubation on acetate plus BES medium with vari­ous sulfur compounds. It is shown that sulfide production and acetate utilization was most efficient in media con­taining sulfate. Reduction of sulfite was incomplete and dithionite reduction could not be demonstrated. Sulfite in concentration of 1000 mg Sil seemed to inhibit sulfite reduction. The medium pH in this experiment remained at 7 in all test bottles during incubation. A significant frac­tion of sulfide that was produced was evolved to the gas. However, this fraction was not measured so a mass balance was not attemped.

7

6

~7 MB/AC :::> S u.. ,J, ::'6

C> .... Ol

.9

~7 SRB LAC. ::J r 0 U

OJ6

..c

. ~ 5 >

7

6

8 12 16 20 43

TIME (Days) Fig. 2. Viable counts of MB growing on formate plus hydrogen plus carbon dioxide and on acetate medium and of SRB on lac­tate and hydrogen plus ~etate plus carbon dioxide medium dur­ing anaerobic degradation of synthetic wastewater in the pre­sence of different sulfur compounds. Symbols as in Fig. 1.

----.oJ -O"l §

C

~ {:200 .- u ~::::J ~ -g 100

L­a.

aJ_ c-' ~§ 30 -f- C (!J 0

E t; 20 aJ ::::J >""0 -f-O cI 0.10 ::::J E ::::J

LJ 2 4 6 8 10 12

TIME (Days) Fig. 3. Methane and sulfide production from methanol by inoculum A. Symbols: (0), 800 mg sulfate SII

(L>.), 800 mg sulfate-SII + BES ('7), 800 mg sulfate-SII + methanol (0), 800 mg sulfate-SII + methanol + BES

c: 0- 60 -,----------,-..,

OJ:';::~ "1:J U Ol LcO 4= ::J E -"1:J- 20 ::Jo

(/,)L-0-OJ 30 §::=­

.r:. E 1Jjc 20 E·Q

4-QJU

. ~-5 10 "Be :50.. E 3 4 B 12 16

TIME (Days)

Fig. 4. Effect of different concentrations of methanol in acetate­methanol media on methane and sulfide production by inoculum B. Symbols: (e), acetate (3 g CODII);

(0), acetate (3 g COD/I) + sulfate (1 g SII); (L>.), acetate (3 g CODIl) + methanol (0.1 g CODII) + sulfate (1 g SII); (0), acetate (3 g CODII) + methanol (1.0 g COD/I) + sulfate (1 g SIl)

Anaerobic Treatment of Sulfur Rich Wastewater 205

Table 1. Utilization of acetate and reduction of sulfate, sul­fite, and dithionite by inoculum B in the presence of BES. Incubation time was 31 days

Sulfur added (mg/I)

Found after 31 days of incubation (mg,1)

Sulfide-S Sulfate-S Acetate!

Sulfate-S 100 500

1000 100 500

1000

5 27

210 450

33 92 5

2 8

11 50 ND ND ND ND ND ND

1775 1605

705

Sulfite-S 90

1595 1360 1565 1575 1635 1630

Dithionite-S 100 500

1000

12 5 5

1 Initial acetate concentration was 1900 mg,1 ND = not determined

Discussion

In reactor studies 20 to 50% of the sulfite from a sulfite evaporator condensate is reduced to sulfide (Eis et aI., 1983; Frostell, 1984), which is what we observed here. Kroiss and Plahl-Wabneq (1983) reported that sulfide tox­icity in anaerobic wastewater treatment decreased with an increasing ratio of COD to S concentration. Sulfide inhibi­tion remains insignificant, if COD/S04 = ratio of the wastewater is over 10 gig (Lettinga et aI., 1985). With this COD-S04 = ratio H2S stripping by biogas produced is thought to keep the H2S concentration in solution below a toxic level. The COD/S04 = ratio of our synthetic waste­water was 1.6 and in other media used in this study it varied from 1 to 1.3. Our results showed that inhibition of methane production in synthetic wastewater was rather due to the initial oxidation state of sulfur (sulfite, dithion­ite) than the sulfide produced during incuBation. The results demonstrated also that the composition of waste­water COD has an influence on methane production and sulfate reduction. The evaluation of sulfide inhibition in anaerobic treatment should rather be based on biogas and sulfate reduction potentials of wastewater constituents than on COD/S04= ratios.

Thiosulfate, dithionate, tritionate, and tetrathionate ions are formed during the dissimilatory of sulfite to sul­fide by Desulfovibrio (postgate, 1984). Dissimilatory reduction of partially oxidized sulfur compounds such as sulfite, thiosulfate, and tetrathionate is not confined to SRB (Pfennig et aI., 1981). In the present experiments no sulfide production could be demonstrated from dithionite in synthetic wastewater or acetate plus BES medium. The results indicated that dithionite may persist in anaerobic environments polluted with mechanical pulping effluents.

Our results gave evidence that sulfide was produced from sulfate when methanol served as the only electron donor and methane fermentation was inhibited with BES. Klemps et al. (1985) isolated two Desulfotomaculum species growing with methanol as the sole source of energy. The utilization of methanol by many pure cultures of Desulfovibrio could not be confirmed (Postgate, 1984).

The methanol utilization by SRB with the failure of dithionite reduction in our experiments suggest that con­ventional Desulfovibrio may not be involved but rather one of the Desulfotomaculum species similar to those iso­lated by Klemps et al. (1985). However, some Desulfovib­rios have also been shown to degrade methanol slowly when pregrown with other substrates (Braun and Stolp, 1985; Nanninga and Gottschal, 1987). Smith and Mah (1978) observed that methanol was a preferred energy source and regulated the degradation of acetate by Methanosarcina barkeri. On the other hand, anaerobic sludge is capable of simultaneously fermenting volatile fatty acids and methanol into methane (Lettinga et al. 1979). Genthner et al. (1981) isolated a bacterium, Eubac­terium limosum, from rumen and sewage sludge that can oxidize methanol to acetate. The methanol utilization pat­tern of our sludge in the presence of BES could be the oxidation of methanol to acetate by bacteria similar to E. limosum and followed by acetate oxidation by SRB or direct utilization of methanol by SRB. Methanol was shown to stimulate methane production but not sulfate reduction in anoxic marine sediments (Oremland et aI., 1982). Methanol oxidizing SRB may be more common than hitherto assumed since few studies have been con­ducted in methanol rich environments. Thermodynamic considerations support this assumption. The standard free energy of the reaction 1/6 CH30H + 118 S04 = + 3/16 H+ ~ 1116 H2S + 1116 HS- + 113 H20 + 1/6 CO2 is -143.4 kJ/mol substrate while in the methanogenic reaction 4 CH30H ~ 3 CH4 + HC03- + H+ + H20 it is -77.7 kJI mol methanol.

Conventional solutions for sulfur associated problems in anaerobic digestion have been the dilution of wastewa­ter before treatment or precipitating sulfide by iron salts or to chemically oxidize H2S. Interactions between methanogenesis and sulfate reduction in sediments are likely to be altered by metals (Capone et aI., 1983). Molybdate ions have been shown to specifically inhibit sulfate reduction in sediments (Oremland and Taylor, 1978). Our recent studies indicate that molybdate inhibi­tion of sulfate reduction cannot be applied in wastewater

206 ]. A. Puhakka, ]. F. Ferguson, M. M. Benjamin, and M. Salkinoja-Salonen

treatment due to its adverse effects on methane production (Puhakka et al., 1988). Preoxidation of partly oxidized forms of sulfur in wastewaters may reduce inhibition and sulfur reduction in anaerobic treatment. Wastestreams which contain non or uncompetitive substrates (that favor methane production over sulfur reduction) should be selected for anaerobic treatment.

Acknowledgements. The work was financially supported by Valle Scandinavian Exchange Program (University of Washing­ton) and the Academy of Finland. We thank Prof. John R. Post­gate for critical review of the manuscript.

References

Abram, J. W., Nedwell, D. B.: Inhibition of methanogenesis by sulfate reducing bacteria competing for transferred hydrogen. Arch. Microbiol. 117, 89-92 (1978)

Badziong, W., Thauer, R. K., Zeikus, J. G.: Isolation and charac­terization of Desulfovibrio growing on hydrogen plus sulfate as the sole energy source. Arch. Microbiol. 116, 41-49 (1978)

Benjamin, M. M., Woods, S. L., Ferguson, J. F.: Anaerobic toxic­ity and biodegradability of pulp mill waste constituents. Water Res. 18, 601-607 (1984)

Braun, M., Stolp, H.: Degradation of methanol by a sulfate reducing bacterium. Arch. Microbiol. 142, 77-80 (1985)

Brune, G., Schoberth, ~~. M., Sahm, H.: Anaerobic treatment of an industrial wastewater containing acetic acid, furfural and sulphite. Proc. Biochem. 16,20-35 (1982)

Capone, D. G., Reese, D. D., Kiene, R. P.: Effects of metals on methanogenesis, sulfate reduction, carbon dioxide evolution, and microbial biomass in anoxic salt marsh sediments. Appl. Environ. Microbiol. 45, 1586-1591 (1983)

Eis, B. J., Ferguson, J. F., Benjamin, M. M.: The fate and effect of bisulfite in anaerobic treatment. J. Water Pollut. Control Fed. 11,1355-1365 (1983)

EPA.: Methods for chemical analysis of water and wastes. EPA-600 4-79-020. Cincinnati, OH 45268, U.S. Environmental Protection Agency 1979

Ferry, J. G., Smith, P. H., Wolfe, R. S.: Methanospirillum, a new genus of methanogenic bacteria, and characterization of Methanospirillum hungatii sp. nov. Int. J. System. Bact. 24, 465-469 (1974)

Frostell, B. : Anaerobic-aerobic pilot scale treatment of a sulphite evaporator condensate. Pulp Paper Can. 85, 80-87 (1984)

Genthner, B. R. S., Davis, C. L., Bryant, M. P.: Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol-and Hz-COz-utilizing species. Appl. Environ. Mic­robiol. 42, 12-19 (1981)

Hungate, R. E.: A roll tube method for cultivation of strict anaerobes. Meth. Microbiol. 3 B, 117-132 (1969)

Khan, A. W., Trottier, T. M.: Effect of sulfur-containing com­pounds on anaerobic degradation of cellulose to methan by mixed cultures obtained from sewage sludge. Appl. Environ. Microbiol. 35, 1027-1034 (1978)

Klemps, R., Cypionka, H., Widdel, F., Pfennig, N.: Growth with hydrogen, and further physiological characteristics of Desul­fotomaculum species. Arch. Microbiol. 143,203-208 (1985)

Kroiss, H., Plahl-Wabneg, F.: Sulfide toxicity with anaerobic wastewater treatment. In: Proc. European Symposium on Anaerobic Waste Water Treatment, Noordwijkerhout, Netherlands (1983)

Lapage, S. P., Shelton, J. E., Mitchell, T. G.: Media for the maintenance and preservation of bacteria. Meth. Microbiol. 3 A, 120 (1970)

Lettinga, G., van der Geest, A ., Hobma, S., van der Laan, J.: Anaerobic treatment of methanolic wastes. Water Res. 13, 725-737 (1979)

Lettinga, G., de Zeeuw, W., Hullshoff Pol, L., Wiegant, W., Rinzema, A.: Anaerobic wastewater treatment based on biomass retention with emphasis on the UASB-process, p. 279-301. In: Anaerobic digestion 1985. Proc. of the fourth Int. Symposium on Anaerobic Digestion, Guangzhou, China, November 11-15, 1985

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, 1373-1379 (1982)

Mah, R . A., Smith, M. R.: The methanogenic bacteria, pp.948-977. In: The Procaryotes (Starr, M. P., eds.). Berlin, Springer Verlag 1981

McCarty, P. L.: Anaerobic waste treatment fundamentals. Part III: Toxic materials and their control. Pub I. Wks. 91-94 (1964)

Nanninga, H. J., Gottschal, J. G.: Properties of Desulfovibrio carbinolicus sp. nov. and other sulftate reducing bacteria iso­lated from an anaerobic purification plant. Appl. Environ. Microbiol. 53, 802-809 (1987)

Oremland, R. S., Taylor, B. F.: Sulfate reduction and methanogenesis in marine sediments. Geochim. Cosmochim. Acta 42,209-214 (1978)

Oremland, R. S., Marsh, L. M., Polcin, S.: Methane producnon and simultaneous sulphate reduction in anoxic salt marsh sedi­ments. Nature (Lond.) 296, 143-145 (1982)

Orion.: Orion Publication 94-16M10811, Orion Research, Inc., Cambridge, MA 02139 (1980)

Owen, W. F., Stuckey jr, J. B., Young, L. Y. and McCarty, P. L.: Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res. 13, 485-492 (1979)

Pfennig, N., Widdel, F., Triiper, H. G.: The dissimilatory sulfate reducing bacteria, pp. 926-940. In: The Procaryotes (Starr, M. P. et aI., eds.). Berlin, Springer Verlag 1981

Postgate, J. R. (ed): The Sulphate-Reducing Bacteria, 2nd ed., Cambridge, Cambridge University Press 1984

Puhakka, J. A., Rintala, J. A., Vuoriranta, P.: Influence of sulfur compounds on biogas production from forest industry waste­water. Water Sci. Tech. 17,281-288 (1985)

Puhakka, J. A., Ferguson, J. P., Benjamin, M. M., Salkinoja­Salonen, M.: Effect of molydate ions on methanation of sulfate rich wastewater. (submitted for publication) .

Rantala, P., Luonsi, A. (eds .): Anaerobic treatment of forest industry wastewaters. Water Sci. Techn. 17, 326 p. (1985)

Smith, M. R., Mah, R. A:.: Growth and methanogenesis by Methanosarcina strain 227 on acetate and methanol. Appl. Environ. Microbiol. 36, 870-879 (1978)

Jaakko Puhakka, Department of Civil Engineering, Water and Environmental Engineering, Tampere University of Technology, P. O. Box 527, SF-33101 Tarnpere, Finland