~ Pergamon

PH: 50273-1223(97)00714-2

War. Sci. Tech. Vol. 36, No. 12, pp. 143-150,1997.© 1997 IAWQ. Published by Elsevier Science Ltd

Printed in Great Britain.0273-1223/97 $17·00 + 0·00

CONTROL OF SULFATE REDUCTION BYMOLYBDATE IN ANAEROBICDIGESTION

Shuzo Tanaka* and Young-Ho Lee**

*Department o/Civil Engineering, Meisei University, Hino-shi, Tokyo 191-8506,Japan** Hyundai Institute o/Construction Technology, Goosung-myun, Yongin-si,Kyunggi-do 449-910, Korea

ABSTRACT

Control of sulfate reduction by adding molybdate was investigated to enhance the methane production under batch andcontinuous operation in the anaerobic digestion of a sulfate-rich lysine wastewater. In phase 1 of the continuousoperation, four anaerobic filters were fed with the lysine wastewater and then added with molybdate at 1,3,5 and 10mM just after methane producing bacteria (MPB) were completely inhibited by HzS produced by sulfate reducingbacteria (SRB). In phase 2, three anaerobic filters were operated with continuous or intermittent addition of 3 mMmolybdate froni the beginning of operation, including one with no molybdate as a control. Batch experiments revealedthat the sulfate reduction was strongly inhibited and finally ceased by adding 3 mM or more of molybdate, resulting ingreat enhancement of the methane production. In phase 1 of the continuous experiments, all reactors showed thecessation of the methane production when the content of HzS reached 9-10 % in biogas, but the MPB activity wasgradually recovered after initiating the molybdate addition at 3 or 5 mM. The 10 mM dosage of molybdate, however,had an inhibiting effect to MPB as well as SRB, resulting in the accumulation of acetate within the reactor. In phase 2,the control reactor continued to decrease the methane production, and a methane conversion rate was only 3% in thecontrol, while 35 and 10 % in continuously-added and intermittently-added reactors, respectively. Thus, it wasconfirmed that the MPB activity was greatly enhanced under control of the SRB activity by the continuous addition ofmolybdate. Comparing phase 2 with phase 1, addition from the start-up of the process is considered more effective thanaddition after the methane production dropped in the control ofthe sulfate reduction by molybdate.© 1997 IAWQ. Published by Elsevier Science Ltd

KEYWORDS

Anaerobic digestion; hydrogen sulfide; methane production; molybdate; sulfate reduction; sulfate-rich wastewater

INTRODUCTION

Anaerobic digestion has been used successfully as a main or a pre-treatment process to treat various high strengthwastewaters. Though the chemistry of anaerobic digestion has been well established, the anaerobic process isn'teffective for every high strength wastewater due to presence of inhibitory substances. Sulfate would often cause aninstability of the process if it's level in the wastewater was high. The presence of sulfate will encourage growth ofsulfate reducing bacteria (SRB) under anaerobic conditions, resulting in formation of H2S which is toxic especially tomethane producing bacteria (MPB), and also SRB will compete with MPB for available substrates such as Hz andacetate. This results in low methane production and then reduction of an energetic advantage ofthe anaerobic digestion.

Parkin et al. (1990) reported that the presence ofunionized HzS at 60 mg S/L caused irreversible failure in an anaerobicchemostat culture, while Hilton and Oleszkiewicz (1988) showed that unionized H2S upto 200 mg S/L could be

143

144 S.TANAKA and Y.-H. LEE

tolerable for MPB. These results and other studies revealed that a toxic level of unionized HlS was between 50-200 mgSIL to substrate utilization and methane production. It has also been reported that sulfide is toxic to SRB. Hilto~ andOleszkiewicz (1988) showed that dissolved sulfide (DS=IUS+HS-+S:") inhibited the sulfate reduction but fuS did notwhen OS levels were high and IUS levels low at high pH. On the other hand, sulfide is one of essential nutrients forgrowth of MPB and SRB, and this makes it complicate to maintain optimal nutrient conditions. The optimal level ofsulfide reported by the previous studies varies from 1-25 mg SIL (Parkin et aI., 1990). Thus, it is important to maintainthe level of sulfide minimum for bacterial growth without inhibition of methanogenesis in order to successfully operatethe anaerobic digestion process of sulfate-rich wastewater.

Various studies have been done to maintain a low level of sulfide in the anaerobic system and some of them are: dilutionof wastewater (Kroiss and Plahl-Wabnegg, 1983); addition of metals such as iron to remove sulfide (Braun and Huss,1982; Frostell, 1982); use of a multistage reactor where the sulfate reduction is limited to acidogenic phase (Reis et al.,1988); stripping of sulfide from a gas recirculation system (Samer, 1990) and addition of alkali to increase pH toconvert toxic HlS to less toxic HS· (Hilton and Oleszkiewicz, 1988; McCartney and Oleszkiewicz, 1991). Mainattempts in these methods seem to reduce the toxicity of sulfide produced rather than to suppress the sulfate reduction.

Analogs of S042' such as Cr042-, Mo042-, W042- and Se042- could be used as a inhibitor for the sulfate reduction bySRB and the approximate order of effectiveness was Cr042- > Mo042- = W042- > Se042- (Taylor and Oremland, 1979).Since chromate (CrOi-) is highly toxic to most of bacteria including MPB, molybdate (Mo042-) has normally been usedto control the sulfate reduction in microbiological experiments because it also selVes as a nutrient for growth of MPB.In treatment of a sulfate-rich wastewater, however, only a few researchers have made attempts to use molybdate tocontrol the sulfate reduction. Hilton and Archer (1988) reported that no fuS was detected in gas phase when themolasses wastewater was treated by an anaerobic filter with continuous addition of 10 mM molybdate but the methaneproduction also decreased. Lo and Liao (1990) showed that both SRB and MPB were inhibited when a fixed-filmreactor treated the bakers yeast wastewater in presence of 2 roM molybdate. Yadav and Archer (1988) also revealed theinhibition of SRB and MPB at 3.2 roM molybdate in an anaerobic filter treating the sulfate-rich wastewater. Asreported by these studies, molybdate might inhibit MPB as well as SRB at levels of 2-10 roM in the anaerobic digestionprocesses. Some other researchers, however, successfully used molybdate to assess the role of SRB without inhibitingMPB in lake and marsh sediments (Smith and Klug, 1981; Lovely and Klug, 1983; Banat et aI., 1983). This imlies thatmolybdate could be used to control the SRB activity In the process of anaerobic treatment if we managed to operate theprocess without inhibiting MPB.

Since little is known on application of molybdate to the control of sulfate reduction and the enhancement of methaneproduction in the anaerobic treatment process of sulfate-rich wastewater, this work was conducted to investigateapplicabilityand operating conditions of this method. Specific objectives are: to obtain the basic infomation on effectsof molybdate addition on the sulfate reduction and the methane production using batch reactors (batch); to find theoptimum dosage of molybdate to control the sulfate reduction and enhance the methane production in the anaerobictreatment of sulfate-rich wastewater using anaerobic filters (phase 1); to evaluate addition methods of molybdate for asuccessful control of the sulfate reduction in the operation of the anaerobic filters (phase 2).

MATERIALS AND METHODS

Industrial wastewater from a lysine (amino acid as a livestock feed) factory was selected for this study, because itcontained a high level of sulfate with 1.67 of COD/S042

- ratio. The lysine wastewater was diluted 40 times for bench­scale experiments using serum bottles and anaerobic filters, and influent characteristics of the filters are summarized inTable 1. In~ulum sludge was obtained from an upflow anaerobic sludge blanket (UASB) reactor fed with the lysinewastewater In our laboratory.UASB granules were disrupted to form uniform suspensions before inoculation.

Table 1. Influent characteristics of anaerobic filters to treat the diluted lysine wastewater

Parameters Concentration Parameters ConcentrationTotal COD (mgIL) 5,000 Total phosphorus (mg PIL) 150Soluble COD (mgIL) 3,100 Sulfate (mg S/L) 1,000Particulate COD (mgIL) 1,900 Total sulfide (mg SIL) 20BOD (mgIL) 2,000 Acetate (mgIL) 200SS (mgIL) 1,700 Propionate (mgIL) 40Total nitrogen (mg NIL) 1,200 pH 6.5

Molybdate in anaerobic digestion 145

A series of batch experiments were primarily conducted using 60 mL serum bottles with 20 mL sludge and 25 mLdiluted lysine wastewater at 37°C. Molybdate (sodium molybdate) ranging from 0 to 50 roM were tested to control theSRB. aci~ty..values of pH were initially adjusted in between 7.0 and 7.2. Strict anaerobic conditions were provided bypurgmg au WIth nitrogen within the solution and then the bottles were immediately sealed by butyl rubber stoppers withaluminum caps. All batch experimentswere duplicated.

Subsequently, two series of continuous experiments were performed using upflow anaerobic filters to treat the dilutedlysine wastewater at 30°C. The anaerobic filter is cylindrical in shape being 105 cm in height and 25 cm in diameterand was packed with plastic fiber contact materials (Bioloop). The empty volume of the filter is 16.8 L and the volumeof Bioloop packed is 1.05 L. In the first phase of the experiments (phase 1), four anaerobic filters were filled with freshseed sludge, and initially operatedwithout molybdate at 2 days of hydraulic retention times (HRT) and then with 1, 3, 5and 10 roM molybdate (sodium molybdate) after a sharp drop of the methane production on day 62 during 100 daysoperation. In the second phase (phase 2), three anaerobic filters were operated at 4 days ofHRT: two with adding 3 roMmolybdate continuously or intermittently from the beginning of operation and one with no molybdate as a control. Theintermittent addition was conducted by adding molybdate every two days. In phase 2, three of the anaerobic filters inphase 1 were used after being seeded with fresh inoculum sludge to replace about half of filter sludge.

Contents of~nstituentsin biogas produced were analyzed using a TCD gas chromatograph with a stainless columnpacked with Porapak Q for CH4 and C02 (GC-15A, Shimadzu) and a FPD gas chromatograph with a glass columnpacked with Shimalite TPA for lliS (GC-8APFp, Shimadzu). Volatile fatty acids (VFA) were analyzed using a Fill gaschromatograph with a glass columnpacked with Carbopack, B-DA 4% Carbowax 20 m (GC-14A, Shimadzu) afterthe samples were filtered through 0.45 J.l.l11 membrane filter and pretreated with oxalic acid. Sulfate and sulfide weremeasured by the turbidimetric method and the idometric method based on Standard Methods, respectively.

RESULTS AND DISCUSSIONS

Effects of molybdate addition in batch reactors. Effects of molybdate addition on sulfate reduction and methaneproduction were investigated primarily by batch reactors containing the lysine wastewater and results after 5 daysdigestion were shown in Fig.l. Organics in the lysine wastewater were almost completely degraded after 5 daysdigestion. When no molybdate was added, sulfate was completely reduced to ffiS, resulting in utilization of about 50%of organics as electron donors in the lysine wastewater, and the remaining organics were converted into methane. Whenmolybdate was added, the sulfate reduction was drastically decreased and it ceased at 3 roM or more addition. As thesulfate reduction was suppressed, the methane production was greatly enhanced and 99% of organics were convertedinto methane at 5mM addition. A 50mM addition of molybdate, however, inhibited the methane production as well as

the sulfate reduction.

100""''$.0 800u'-"

§ 60'+J.I::il~ 40::lCI)

1;j

tl 20.gCIl

00 2 3 5 50

Molybdate added (mM)

• Methane produced • Sulfate reduced 0 Unutilized substrate

Fig.l. Effects of molybdate addition on substrate utilization after 5 days in batch reactors

Effects ofmolybdate addition in phase 1. Four anaerobic filters to continuously treat the lysine wastewat~r in phase 1were initially operated without molybdate and then with 1, 3, 5 an~ ~O roM molybdate after day 62 dunng 100 daysoperation. All reactors showed the strong inhibition of the MPB actIVIty as sulfate was reduced to ffiS before day 62.

146 S.TANAKA and Y.-H. LEE

After starting the molybdate addition, both 3 and 5 roM reactors gradually recovered in activities of methane production,but 1 and 10 roM reactors did not.

The biogas production over the whole period of operation in the 5 roM reactor was shown in Fig.2. Before day 62,methane was actively produced at the beginning of operation but it gradually decreased as the production of H2Sincreased. When the content of IUS reached 5~7 % in the evolved biogas on day 40·50, the methane production began todrop further down and it finally ceased shortly after the content of IUS became 9-10 % in biogas. Based on the Henry'slaw, the 5 % contents of IUS in the gas phase correspond to about 130 mg SIL as total sulfide in the liquid phase.Additionally, the production rate of IUS also began to decrease at levels of 9-10 % H2S in biogas. On the other hand,after day 62 the production of IUS was decreased in presence of molybdate and it ceased on day 74. Since the sulfatereduction was suppressed by molybdate, the methane production was gradually increased, although the MPB activitywas not completely recovered to an initial level during 40 days operation in presence of molybdate.

5 16

14

9080

• CH4

~H2S

70

: With Molybdate (5mM) _

60504030

Without Molybdate

2010o

~.......,2o 6-........--L. L.-.a.-.....L-........--&.---I........L-........:::-..,•• :::X:>-<>-<:>-C..-<JI-Oi-<J 0

100

Elapsed Time (day)

Fig.2. Methane production and hydrogen sulfide content in biogas in phase 1

100

o100908070605040

1500

3500 500

With Molybdate

• 4003000

~

~ 300

i 2500

~ 200CI)

2000

Elapsed Time (day)

• ImM • 3mM • 5mM • IOmMMolybdate~ ImM --<>- 3mM ~ 5mM '**"**i:Jr- IOmM

Fig.3. Effects of molybdate addition after day 62 on sulfate reduction in phase 1

Molybdate in anaerobic digestion 147

Levels of sulfate and sulfide in effluent before and after molybdate addition (day 62) at concentrations of 1-10 mMwere shown in Fig.3. Before day 62, the SRB activity was very high and about half of influent sulfate was reduced toH2S. The levels of total sulfide around day 50, at which the MPB activity was greatly dropping, were 220-300 mg SIL,and they reached 400-450 mg SIL when the SRB activity was inhibited as well. Since the values of pH in effluentaround day 50 were about 7.8, the computed concentrations of undissociated ffiS are 30-40 mg SIL for 220-300 mgSIL total sulfide. After day 62, the concentrations oftotal sulfide in effluent dropped to the levels of influent in presenceof 3-10 mM molybdate, but about 100 mg SIL of total sulfide remained in case of I mM addition. Following theinhibition of the sulfate reduction, the sulfate concentrations in effluent were increased to the influent levels except for ImM addition. These dosages of molybdate to inhibit the SRB acitvity correspond to requirements obtained in the batchreactors explained earlier.

Accumulation of volatile fatty acids in all four reactors was analyzed before and after molybdate addition as shown inFig.4. Acetate accumulated at about 1,300 mgIL during the MPB-inhibited period before day 62 but it significantlydropped by day 70 shortly after initiating the molybdate addition in all reactors. Behaviors of volatile fatty acids afterday 70, however, varied with each dosage of molybdate. In 1 and 10 mM additions, the levels of acetete once wentdown to 400-600 mgIL after day 70 but again went up to the levels of 1,200 mgIL thereafter. The accumulation ofpropionate was observed in the 10 mM reactor only while the acetate level was kept low. In 3 and 5 mM additions,acetate had been kept low at 400-500 mgIL after day 70 and propionate was accumulated at 250-300 mgIL. Weconsider that the maintenance of low levels in acetate is due to utilization by MPB which recovered from the HzStoxicity after the molybdate addition. The accumulation of propionate is probably due to no decomposition by SRBwhose activity was inhibited by molybdate. It seems that the 10 mM dosage of molybdate had an inhibiting effect toMPB as well as SRB, resulting in the accumulation of acetate as shown in Fig.4d).

1400

1200

1000i 800'-"« 600

~ 400

200

o50

-.-Acetate~ Propionate-A- Butyrate

60 70 80 90Elapsed Time (day)

100

1400

1200

1000i 800

~ 600;> 400

200

o50

____ Acetate~ Propionate--..- Butyrate

6070 80 90 100Elapsed Time (day)

a) ImM molybdate addition b) 3mM molybdate addition

1400

1200

-- 1000! 800

« 600

~ 400

200

o50.

____ Acetate~ Propionate-A-Butyrate

6070 80 90Elapsed Time (day)

100

1400

1200

1000i 800

« 600

~ 400

200

o50 60 70 80 90

Elapsed Time (day)100

c) 5mM molybdate addition d) 10mM molybdate addition

Fig.4. Effects of molybdate addition after day 62 on volatile fatty acids (VFA) accumulation in phase 1

148 S.TANAKA and Y.-H. LEE

Evaluatwn of molybdate addition methods in phase 2. To evaluate addition methods of mol~bdate to co~trol thesulfate reduction, three anaerobic filters were operated: two with adding 3 roM molybdate to the influent con~uouslyor intermittently from the beginning of operation and one with no molybdate as a control. The intermittent addition ~asconducted by adding molybdate every two days. In here, three of the anaerobic filters in phase 1 were used after bemgseeded with fresh inoculum sludge to replace about half of filter sludge. The biogas production from these three reactorswas shown in Fig.5.

......- Continuous--+-lnterrnittent---.- Control

1.2

,....,~~cl 0.8§

] 0.6

e'; 0.4'"tlIloas 0.2

oo 10 20 30

Elapsed Time (day)

40 50

a) Biogas production

30

25,-...

20 ~0.......,Cl

15 B8u

10 rI)

:B5

• Control--/:r= Control

4010

~::_ •.-:~:=.~.~.~.-:-.~:-~ 0

50o 20 30Elapsed Time (day)

-.-Continuous • Intermittento Continuous e Intermittent

CH4H2S

80

70,-...

";f? 60.......,

I 50u 40

~ 30:; 20

10

o

b) Methane and hydrogen sulfide contents of biogas

Fig.5. Biogas production when 3roM molybdate were continuously or intermittently added in phase2

Rates of the biogas production were dropped shortly after starting the operation in all three reactors and became almostconstant after day 30 in continuous and intermittent reactors,although the production rates were low in all reactorscompared with those before the MPB activity dropped in phase 1. The control reactor continued to decrease the biogasproduction and the methane content in the gas during the whole periods of operation, and it produced the biogas at only0.1 Lid with 40 % methane and 11 % H2S on day 50. On the other hand, the biogas was constantly produced at 0.6-0.7Lid with 70-75 % methane and no H2S when molybdate was continuously added, and at 0.4-0.5 Lid with 60-65 %methane and 4-5 % H2S when intennittently added. Comparing phase 2 with phase 1, addition from the start-up of theprocess is considered more effective than addition after the methane production dropped in the control of the sulfatereduction by molybdate.

Effluent characteristics of continuously-added and intermittently-added reactors at steady state were summarized inTable 2, including results on day 50 in the control reactor. No big differnce was observed in the total COD removal

Molybdate in anaerobic digestion 149

among three reactors, which were 42-45 %. This is because half of COD removed were particulate, which wereremoved by ac~urnulationwithin the reactors. Methane conversion rates, however, were 35, 10 and 3% as percents ofmethane COD m solu?le COD removed in continuous,intermittent and control reactors, respectively. Also, levels ofsulfate and sulfide venfied the suppression of the sulfate reduction when molybdate was added. Thus it was confirmedthat the MPB activity was greatly enhanced under control of the SRB activity by the continuous addition of molybdate.

Table 2 Effluent characteristics of continuouly-added and intermittently-added reactorswith 3 mM molybdate at steady state in phase 2

Parameters ContinuousTotal COD (mgIL) 2,753Soluble COD (mgIL) 2,090Particulate COD (mg/L) 663Sulfate (mg SIL) 900Total sulfide (mg SIL) 80Acetate (mg/L) 820Propionate (mg/L) 220Butyrate (mg/L) 80plI 7.6Biogas production (Lid) 0.65methane content (%) 73I-hS content (%) 0Total COD removal (%) 45Methane conversion (%)** 35* Effiuent characteristics with no molybdate on day 50.**Percent ofmethane COD in soluble COD removed.

CONCLUSIONS

Intermittent2,7552,090

665750185

1,00017070

7.70.45

634.54510

Control*2,9202,000

920570350

1,2404550

7.90.10

40Il.l

423

Applicability and addition methods of molybdate to control the sulfate reduction were evaluated in the anaerobicdigestion of the sulfate-rich wastewater. Batch experiments revealed that the sulfate reduction was strongly inhibitedand finally ceased by adding 3 mM or more of molybdate, resulting in great enhancement of the methane production.All continuous reactors showed the cessation of the methane production when the content of lI2S reached 9-10 % inbiogas, but the MPB activity was gradually recovered after initiating the molybdate addition at 3 or 5 mM. The 10 mMdosage ofmolybdate, however, had an inhibiting effect to MPB as well as SRB, resulting in the accumulation of acetatewithin the reactor. In the study of molybdate addition methods, a methane conversion rate was only 3% in the control,while 35 and 10 % in continuously-added and intermittently-added reactors, respectively. Thus, it was confirmed thatthe MPB activity was greatly enhanced under control of the SRB activity by the continuous addition of molybdate.Addition from the start-up of the process is considered more effective than addition after the methane productiondropped in orde! to control the sulfate reduction by molybdate.

ACKNOWLEDGEMENT

This study was supported by the Japan International Cooperation Agency (JICA) through Asian Institute of Technologyand the Grant-in-Aid for Scientific Reseach (B) of the Ministry of Education, Science, Sports and Culture, Japan. We

wish to express our gratitude to them.

REFERENCES

Banat, I.M., Nedwell, D.B. and Balba, M.T. (1983). Stimulation ofmethanogenesis by slurries of saltmarsh sedimentafter the addition ofmolybdate to inhibit sulfate reducing bacteria. J. General Microbiol., 129, 123-129.

Braun, R. and lIuss, S. (1982). Anaerobic digestion of distillery effluents. Process Biochem., 17 (4),25-27.Frostell, B. (1982). Anaerobic fluidized bed experimentation with a molasses wastewater. Process Biochm., 17 (6), 37-

40.lIilton, M.G. and Archer, D.B. (1988). Anaerobic digestion of a sulfate-rich molasses wastewater: Inhibition of

150 S.TANAKA and Y.-H. LEE

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Lovley, D.R. and Klug, M.J. (1983). Sulfate-reducers can outcompete methanogens at fresh water sulfateconcentrations. Appl. Environ. MicroblOl., 45, 187-192.

McCartney, D.M. and Oleszkiewicz, JA. (1991). Sulfide inhibition of anaerobic degradation of lactate and acetate. Waf.Res., 25, 203-209.

Parkin, G.F., Lynch, N.A., Kuo, W., Keuren, E.L.V. and Bhattacharya, S.K. (1990). Interaction between sulfatereducers and methanogens fed acetate and propionate. J. Wat. Poll. Cont. Fed., 62 (6), 780-788.

Reis, M.A.M., Goncalves, L.M.D. and Carrondo, M.J.T.(1988). Sulfate reduction in acidogenic phase anaerobICdigestion. Wat. Sci. & Tech., 20 (11/12), 345-351.

Samer, E. (1990). Removal of sulfate and Sulphite in an anaerobic trickling filter. Wat. Sci. & Tech., 22 (1/2), 395-404.Smith, R.L. and Klug, M.J. (1981). Electron donors utilized by sulfate reducing bacteria in eutrophic lake sediment.

Appl. Environ. Microbiol., 42, 116-121.Taylor, B.F. and Oremland, R.S. (1979). Depletion of adenosine triphosphate in Desulfovibrio by oxyanions of Group

VI elements. Current MlcroblOl., 3, 101-103.Yadav, VK. and Archer, D.B. (1989). Sodium molybdate inhibits sulfate reduction in the anaerobic treatment of high­

sulfate molasses wastewater. Appl. Microbiol. & Biotech., 31, 103-106.