SULFIDE-INDUCED INHIBITION

OF ANAEROBIC DIGESTION

By Bar ry L. Hilton1 and Jan . A. Oleszkiewicz,2 Member , ASCE

ABSTRACT: Batch anaerobic digestion studies of the effects of pH and sulfide concentration upon lactose utilization, acetate utilization, and sulfate reduction us­ing lactate as a carbon source were performed using 100 ml glass syringes. In­creased concentrations of un-ionized H2S inhibited lactose utilization and acetate utilization whereas total sulfide concentration, more than un-ionized H2S, inhibited sulfate reduction. Increased retention time was required to carry lactose and acetate utilization to completion in the presence of high concentrations of TS. Almost 90% utilization of lactose at undissociated H2S in excess of 900 mg/L was attained at a retention time of 228 hr. Acetate utilization was 100% complete after 74 days at 175 mg/L-H2S and deteriorated rapidly to zero at higher H2S concentrations. A mechanism is proposed whereby the carbon flow can be diverted from sulfate re­duction to methanogenesis in spite of the presence of a high concentration of sul­fate.

INTRODUCTION

The formation of hydrogen sulfide in anaerobic reactors is the result of the reduction of oxidized sulfur compounds and of the dissimilation of sul­fur-containing amino acids such as cysteine. In anaerobic reactors, sulfur reduction is performed by two major groups of sulfate reducing bacteria (SRB): (1) Incomplete oxidizers which oxidize compounds such as lactate to acetate and C 0 2 (Eq. 1); and (2) complete oxidizers (acetoclastic SRB) which com­pletely oxidize acetate to C 0 2 and HCO^ (Eq. 2). Both groups utilize hy­drogen for sulfate reduction:

2 CHjCHOHCOO" + S O r -* 2 CH3 COCT + 2 CO, + S2~ (1)

CH3COO" + SO^" -» H 20 + C0 2 + HC03~ + S2~ (2)

Generated sulfide (denoted total sulfide—TS) consists of three species: H2S, HS~ and S~2. The dissolution of H2S in water forms the equilibrium system

H2S «* H+ + HS" ^ 2H+ + S2" . (3)

The decimal fraction of un-ionized H2S in solution can be determined from the following equation:

H2S = [1 + 1.02 * 10(pH-7)]_1 (4)

The percentage of un-ionized H2S drops from 90% at pH 6.0 to 50% at pH

'Dept. of Civ. Engrg., Univ. of Tennessee, Knoxville, TN; formerly Grad. Res. Asst., Envir. Engrg. Div., Dept. of Civ. Engrg., Univ. of Manitoba, Winnipeg, Canada R3T 2N2.

2Assoc. Prof., Envir. Engrg. Div., Dept. of Civ. Engrg., Univ. of Manitoba, Win­nipeg, Canada R3T 2N2.

Note. Discussion open until May 1, 1989. To extend the closing date one month, a written request must be filed with the ASCE Manager of Journals. The manuscript for this paper was submitted for review and possible publication on February 23, 1988. This paper is part of the Journal of Environmental Engineering, Vol. 114, No. 6, December, 1988. ©ASCE, ISSN 0733-9372/88/0006-1377/S1.00 + $.15 per page. Paper No. 23017.

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7.0 to 10% at pH 8.0. This variation is most significant in anaerobic treat­ment since the pH range of anaerobic reactors is maintained between pH 6.0 and 8.0 with the generally accepted optimal pH for methane production being between pH 6.8 and 7.5. Furthermore, for experimental purposes, by ad­justments in pH, it is possible to study the relationship between different concentrations of un-ionized H2S and different aspects of anaerobic treatment such as lactose uptake, sulfate reduction, and acetate uptake.

Studies have shown that methanogenic bacteria can tolerate sulfide con­centrations up to 1,000 mg/L TS (Mountfort and Asher 1979; McKinney, personal communication, 1986). Kroiss and Plahl-Wabnegg (1983), in a study of flocculant sludge, found a complete loss of methane production at 200 mg/L of un-ionized H2S. The decrease in methane production coincided with a decrease in carbon removal and an increase in the concentration of volatile fatty acids (VFA). When the H2S concentration increased above 200 mg/L, the formation of VFA decreased. Both Kroiss and Plahl-Wabnegg (1983) and Koster et al. (1986) found inhibition of methanogenesis at H2S concen­trations as low as 50 mg/L. This is only two to six times the concentration of 8-22 mg/L TS required for optimum conditions for methanogenic bac­teria (Wellinger and Wuhrmann 1977; Ronnow and Gunnarson 1982). It ap­pears that toxicity of sulfide depends on the nature of substrate, type of biomass, and primarily type and concentration of a given sulfide species.

PURPOSE AND SCOPE

Presently, there is a general lack of understanding of the inhibition of methanogenesis by the various species of sulfide and by the SRB. Prime factors in understanding the sulfide inhibition are competition for hydrogen at various trophic levels, dynamic population shifts induced by substrate preference by a domineering group (e.g., preference for lactate by incom­pletely oxidizing SRB), and different levels of tolerance to the H2S-HS~ system by the various bacteriological groups involved in the system, with and without the accompanying population shifts.

Preceding continuous-culture studies (Oleszkiewicz and Hilton 1986; Hil­ton and Oleszkiewicz 1987a, 1987b) left unanswered questions concerning the effects of total sulfide and un-ionized hydrogen sulfide upon lactate uti­lization, methanogenesis, and sulfate reduction.

This paper presents the results of a study of the effects of a wide range of sulfide concentrations upon three processes occurring during the anaerobic treatment of high sulfate wastes: (1) Utilization of a disacharide such as lactose; (2) acetate uptake or methanogenesis; and (3) carbon flow under conditions of high sulfate concentration and varying concentrations of un­ionized H2S. It was expected that sulfide toxicity to all three groups of bac­teria, the lactose utilizing, acetate utilizing, and sulfate reducing bacteria, would be due to the increased concentration of un-ionized H2S, and not the concentration of total sulfide (TS).

EQUIPMENT AND PROCEDURES

All experiments were conducted in 100 ml Perfectum™ glass syringes with fitted plungers in accordance with the technique described by Sobkowicz and Klemm (Badger Engrs., Inc., Cambridge, Mass., unpublished paper, 1986).

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TABLE 1. Medium Used in Experiment 2

Compound (1)

KH2P04

NH„C1 CaCl2 • 2H20 MgCl2 • 6H20 KC1 NaCl Yeast extract Acetic acid Trace elements

g/L (2)

0.40 0.60 0.30 0.80 0.60 0.40 0.40 2.00 2.00 ml/L

Specially designed stainless steel tips were machined such that one end fitted the Luer-Loc™ tips of the syringes and the other end accepted rubber gas stop septa. The plungers moved easily permitting maintenance of atmo­spheric pressure. The syringes were incubated in a 35° C water bath.

Three separate experimental series were run. In Experiment 1, the effects of added sulfide on lactose utilization were studied. In Experiment 2, the effects of sulfide on acetate uptake (equivalent to methanogenesis) were as­sessed. In Experiment 3, the effects of added sulfide, with lactate as a carbon source, on the reduction of sulfate were studied. Each experiment was run in three series: at initial pH = 6.0, 7.0, and 8.0. The initial pH adjustment was made using NaOH and HC1. There was no pH adjustment during in­cubation.

The liquid mixture for individual experiments was prepared as follows. Inocculum (sludge) was collected from an appropriate active breeder reactor fed whey (for Experiment 1), acetate (Experiment 2), or lactate and sulfate (Experiment 3). Five milliliters of inocculum were mixed with 50 ml of the organic media prepared, that is, in Experiment 1 a stock solution of pow­dered whey (2,000 mg/L equivalent to 800 mg/L total organic carbon (TOC), in Experiment 2 a stock medium as in Table 1 with trace elements as in Table 3, and in Experiment 3 a stock medium as in Tables 2 and 3. Deion-

TABLE 2. Medium Used in Experiment 3

Compound (1)

KH2PO„ NH4C1 CaCl2 • 2H2

MgCl3 • 6H20 KC1 NaCl Yeast extract Lactic acid Na2S04

Trace elements

g/L (2)

0.40 0.60 0.30 0.80 0.60 0.40 0.40 2.20 3.60 2.00 ml/L

Note: After Medium G (Postgate 1984).

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TABLE 3. Trace Elements Solution

Compound (1)

FeCl2-4H20 H3B03

MnCl2 • 4H20 CoCl2 • 6H20 ZnCl2

NiCl2 • 6H20 CuCl2 • 2H20 NaMo04 • 2H20

g/L (2)

1.50 0.06 0.10 0.12 0.07 0.025 0.015 0.025

ized water was added to make a 100 ml solution. All syringes, in all three experiments, were filled to 60 ml of their volume. All syringes had a starting TOC concentration of 400 mg/L, except in Experiment 3 where the TOC was 440 mg/L.

Each series, within any of the experiments, had sodium sulfide added in order to arrive at concentrations of 0, 50, 100, 250, and 1,000 mg/L TS. Gas analyses were performed on a Gow-Mac 550 gas chromatograph (GC) equipped with a thermal conductivity detector, stainless steel column, and Poropak Q packing. Helium was the carrier gas. The VFA were analyzed On a Gow-Mac 750 GC equipped with a flame ionization detector and bo-rosilicate glass column packed with 80/100 mesh Chromosorb 101.

TOC analyses were performed using a Dohrmann DC-80 TOC Analyzer equipped with an UV detector, automatic sampler, and an integrator. Sulfide analyses were performed using an Orion™ specific ion probe. Calibrations were made daily. Sulfate analyses were performed on a Technicon Autoan-alyzer in accordance with the American Public Health Association (APHA) (1985). Lactose was analyzed on a Waters high pressure liquid chromato­graph (HPLC) equipped with a Biorad HPX-85H column and a Biorad re­fractive index detector. Total and volatile nonfilterable residue and all other analyses were performed in accordance with the American Public Health Association (APHA) (1985).

RESULTS

Effects of Sulfide upon Lactose Utilization—Experiment 1 Reconstituted powdered whey containing 68% lactose was the carbon and

nutrient source in previous experiments (Oleszkiewicz and Hilton 1986; Hil­ton and Oleszkiewicz 1987a, 1987b). Lactose (a major component of milk solids and whey) is a disacchaiide, galactose-b-l,4-glucose. One mole of lactose is dissimilated into two moles of glucose-6-phosphate during gly­colysis (Zubay 1984). In this batch study, the rate of utilization or uptake of lactose was used as a measure of glycolysis.

Lactose utilization or uptake versus time at varying concentrations of so­dium sulfide is shown in Fig. 1. The syringes were run in three series at initial pH values of 6.0, 7.0, and 8.0. The most rapid uptake of lactose was observed at pH 6.0 in the absence of sulfide. The moment sulfides were added, lactate utilization began to suffer in the low-pH syringes.

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0 2 4 6 8 10 • 12 Time, hours

FIG. 1. Lactose Utilization versus Time at Various Initial pH Values; Legend Re­fers to Initial Sulfide Concentrations

The induction period (period before uptake commenced) decreased with increases in the pH at higher concentrations of TS. At lower TS the induction was actually shorter at lower pH. At 1,000 mg S2~/L, lactose uptake was completed after 9 hr at pH 8.0, and 24 hr at pH 7.0 (not shown in Fig. 1). The uptake (at 1,000 mg/L TS) remained incomplete after 200 hr at the lowest pH series (not shown in Fig. 1). The rate of lactose uptake (lactose removed per unit time) gradually decreased with increasing concentrations of sulfide. It was concluded that sulfide inhibits and retards the metabolism of the microbes which utilize lactose, presumably the fermentative or gly­colytic microorganisms.

Without added sulfide, the rate of lactose uptake increased as the pH de­creased, indicating the expected behavior of acidogenic microorganisms (Fig. 1, points for TS = 0). The lowest concentration of sulfides (100 mg/L) already resulted in a decrease of the rate of lactose uptake with the decrease in pH. At 100 mg/L TS full utilization of lactose was accomplished after 8 hr at pH 6.0, 7 hr at pH 7.0, and 6 hr at pH = 8.0. Since concentration of the un-ionized H2S decreases with increasing pH (Eq. 4), these results show that sulfide toxicity to the process of lactose uptake could be mini-

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140

120

-a <D N

!

o 4 h r s

+ 24 h r s

A 8 h r s

« 228 h r s

* 12 h rs

0 0 # ^ H ^

£00 400 600 800 Concen t ra t i on o f H2S. mg/L

FIG. 2. Lactose Utilization versus Un-lonized H2S

1000

mized by increasing the reactor pH. It follows from Fig. 1 that the increase to pH 7.0 is adequate unless the TS concentration exceeds 500 mg/L.

Lactose uptake was plotted versus calculated concentration of H2S in Fig. 2. The data are from all three pH series. After 4 hr, 20-30% uptake of the lactose was observed up to 250 mg/L H2S. There was little difference in lactose uptake between 12 hr and 24 hr when the un-ionized H2S was above 250 mg/L. However, after 228 hr, 100% uptake was achieved at 500 mg/ L H2S and 80% at 950 mg/L H2S.

The latter finding demonstrates that the completion of lactate utilization was achieved by increasing the retention (contact) time. This compensated for the lowered metabolic rate of the microorganisms in the presence of high concentrations of un-ionized H2S. The effects of time on lactate uptake could also be explained by the gradual population shift or adaptation. The batch nature of these tests does not allow for distinguishing between population shift and lowered metabolic rates.

Kroiss and Plahl-Wabnegg (1983) noted a decrease in the VFA concen­tration and a decrease in the COD removal in digesters in which the un­ionized H2S was greater than 200 mg/L. In the presence of high concen­trations of H2S, the complex carbohydrates fed to an anaerobic reactor will not be as readily broken into the simplest fatty acids which are the source of energy and carbon for either sulfate reducing bacteria (SRB) or meth-anogens (MPB). If lactose utilization is inhibited, process failure may occur due to substrate limitation (precursor inhibition) to the succeeding bacterial groups (SRB, MPB).

The data in Figs. 1 and 2 suggest that maintenance of long HRT or sig­nificantly larger SRT than HRT, at slightly alkaline pH, should decrease

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sulfide toxicity to the lactose utilization processes (acidogenesis) provided that the undissociated sulfide concentration stays within 500 mg/L H2S.

Effects of Sulfides upon Methanogenesis—Experiment 2 The activity of methanogenic bacteria was measured in terms of acetate

utilization. Acetate utilization versus time for syringes fed acetate and vary­ing concentrations of sodium sulfide at three final pH values of 6.5, 7.4, and 8.0 is shown in Fig. 3. The effects of pH on methanogenesis at TS = 0 followed the expected trend of slight inhibition at low pH = 6.5. There was no inhibition due to pH between series at 7.4 and 8.0. At a final pH of 6.5, acetate utilization was completely inhibited at concentration of 500 mg/L TS (450 mg H2S/L) and above. The induction time was considerably shortened with the increase of pH for all syringes at TS above 150 mg/L. At pH a 7.4, all acetate was utilized on or before day 75 in the presence of 1,000 mg/L TS.

Acetate removal versus the calculated un-ionized H2S is shown in Fig. 4 using the data base from Fig. 3. With increases in time, acetate was com-

pH = 8.0 „ 0 mg/L

t - 50 mg/L " > 5 0 m3A-

L 250 mg/L - 5 0 0 n ^ / L

o 750 mg/L - 1000 mg/L

30 40 90

T i ms, Days

FIG. 3. Acetate Utilization versus Time at Various Final pM Values; Legend Re­fers to Initial Sulfide Concentrations

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100*

-6 80 ffl >

CD

& 60

$ 8 40 <r

+ Day 8

20

\

Day 17 n Day 25 O Day 74

-tP [£>-0 O

W

'^B-50 100 150

Concen t ra t ion o f H2S

FIG. 4. Acetate Utilization versus Un-lonized H2S

200

pletely removed at progressively increasing concentrations of H2S. After 8 days, 100% of the acetate was removed at 20 mg/L H2S, but only 50% of the acetate was removed at 45 mg/L H2S. At day 17, 100% was removed at 60 mg/L H2S and 60% at 90 mg/L H2S. After 25 days, 100% was re­moved at 90 mg/L H2S. After 75 days, 100% was removed at 175 mg/L H2S. The detrimental effects of increased concentrations of un-ionized H2S on methanogenesis resulted in the requirement for a longer retention time (contact time). The level of 200 mg/L H2S, generally assumed as a maxi­mum toxic level severely or completely retarding methanogenesis, could not be compensated by extending the contact time (SRT = HRT in this study). The batch nature of the study did not allow for determination if one was dealing with species adaptation, slowed down metabolism, or population shift towards a more resistant biomass.

Kroiss (personal communication, 1987) has demonstrated, in full scale, uninhibited treatment of citric acid plant effluent in an upflow anaerobic sludge bed (UASB) reactor in the presence of approximately 117 mg/L H2S. Granular sludge in any UASB reactor has SRT well in excess of 50 days. Koster et al. (1986) found that the inhibition of methanogenesis due to total sulfide (TS) concentration was similar at pH 7.0-7.2 and at pH 7.8-8.0. In the research reported here, however, the inhibition of methanogenesis was directly correlated to the pH. It was concluded that methanogens are directly affected by high concentration of un-ionized H2S and that concentration of total sulfides is of lesser significance.

Characteristically, the effects of added sulfides on acetate uptake by meth­anogens were much more severe than on the lactose degradation (acidoge-nesis). Acidogenesis proceeds at much faster rates, and the effects of sulfides on acetate utilizers begins at a much lower threshold.

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0 5 10 15 20 25 30 ' T i m e , Days

FIG. 5. Sulfur Reduction versus Time at Various Final pH Values; Legend Refers to Initial Sulfide Concentrations

Effects of Sulfides upon Sulfate Reduction—Experiment 3 The operation of continuous flow sulfidogenic reactors (Oleszkiewicz and

Hilton 1986; Hilton and Oleszkiewicz 1987a, 1987b) raised questions re­garding the effects of increased concentrations of sulfide upon the sulfate-reducing process. It was shown that incomplete oxidizing sulfate-reducing bacteria were predominant in the reactors fed sulfates and lactate (Hilton 1987). Lactate (Table 2), the preferred carbon source for incomplete oxi­dizing SRB, was used in this experiment.

Sulfur reduction versus time for syringes fed lactate (C0 = 440 mg/L TOC), sodium sulfate (S0 = 533 mg/L S6+), and varying concentrations of sodium sulfide is shown in Fig. 5. The final pH values were 6.5, 7.3, and 8.0 in the individual series. The amount of sulfate reduction decreased with the increases in sulfide concentration, which suggests a decrease in the met­abolic rate of the SRB. This is similar to the conditions observed for lactose uptake and methanogenesis (Experiments 1 and 2) in which increased con­centrations of sulfide directly retarded the rate of metabolism. In the cases of lactose uptake and acetate uptake, inhibition due to TS was alleviated with increased pH values. In the case of sulfate reduction, that was not the

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300

250 Theo re t i caI Max i mum h^4- • y O ' T r ;

50

\ \ " - A — k

0 100 200 300 400 500 600 700 800 900 1000 I n i t i a l S u l f i d e Concen t ra t i on . mg/L

FIG. 6. Sulfur Reduced versus Initial Sulfide Concentration, Carbon Limited Sys­tem—Day 21

case, as is illustrated in Fig. 6 where sulfur reduction was plotted versus the initial concentration of sulfide for day 21.

Sufficient lactate (C0 = 440 mg/L TOC) was fed to achieve a reduction of 222 mg/L sulfur by incomplete oxidizing SRB (based upon the stoichi-ometry of Eq. 1). In other words, this system was carbon limited. If inhi­bition were due to un-ionized H2S rather than TS, more sulfur would have been reduced at pH 8.0 than at pH 6.5 for any given initial TS concentration. The experiment proved, however, that the amounts of sulfate reduced were comparable at all three pH values.

In another experiment, performed as an extension of Experiment 3, a set of syringes was maintained at pH 7.0, with a much higher concentration of lactate (C0 = 6,000 mg/L TOC). This amount of carbon was sufficient to permit the reduction of 2,000 mg Ss+/L (Fig. 7). The experiment was ini­tiated to test the inhibition of SRB by sulfide under unlimited substrate con­ditions. Sulfur reduction and carbon removal were completely inhibited in the syringe containing 1,600 mg S2~/L (not included in Fig. 7). The max­imum sulfur reduction attained was 1,096 mg S6+/L when the initial sulfide (TS) concentration was less than 100 mg/L. Above 150 mg/L TS, a sig­nificant inhibition of sulfur reduction was observed. The data suggested that sulfate reduction would not proceed when the total sulfide concentration was above 1,000 mg/L TS, regardless of the availability of carbon or sulfate.

The value of 150 mg/L TS corresponds to 75 mg/L of un-ionized H2S (pH = 7.0). A similar threshold of inhibition was observed in the studies of methanogenic toxicity. The inhibition patterns in Figs. 6 and 7 are quite similar and appear independent of the amount of sulfur reduction and carbon available.

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i200

1000

CD E

(D

s CD

5

0 100 200 300 400 500 600 700 800 900 1000

I n i t i a l S u l f i d e Concen t ra t i on . mg/L

FIG. 7. Sulfur Reduced versus Initial Sulfide Concentration, Excess Carbon and Excess Sulfur at pH 7.0

400

_l 300 \ CD E

"D

> O E CD 1_

a o

200

100

H ^ "

o-V^

O TOC. pH 6.3

• TOC. pH 7.3

+ TOC. pH 8.0

i r

*T ==**'*r + — • - — +

\

\ \ p ^—m

-v

\ ^ ^ " ' D

O

400

300 ro

"D (D 0 3

200 XI m

or i_ 3

100 3

200 400 600 800 1000 1200 F i n a l S u l f i d e C o n c e n t r a t i o n . mg/L

FIG. 8. Sulfur Reduction and TOC Removal versus Final Sulfide Concentration

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Carbon Utilization in Experiment 3 Sulfur reduction and TOC removal data from Figs. 5 and 6 were plotted

against the final total sulfide concentration (Fig. 8). Each point for sulfur reduction represents an average of sulfur reduced at three pH values (Fig. 5) but at the same final TS concentration.

Sulfur reduction was inhibited with increased concentrations of TS, as was already shown in Figs. 5 and 6, regardless of pH. TOC removal was directly affected by the pH and final sulfide concentration. At 1,100 mg/L TS, only 45 mg/L TOC were removed at a final pH of 6.4, whereas 350 mg/L TOC were removed at pH 8.0. The increased TOC removal at the higher pH was due to increased methane production, which was the highest in the syringes operated at pH 8.0.

It is shown in Fig. 4 that acetate utilization was significantly inhibited by un-ionized H2S, and much less by total sulfide. In Fig. 6, it is shown that sulfate reduction is inhibited more by the increasing concentration of TS than by H2S. This product inhibition provides conditions whereby the carbon flow in an anaerobic reactor can be diverted from sulfur reduction to the produc­tion of methane, even in the presence of a carbon source preferred by the incomplete oxidizing sulfate reducing bacteria (SRB).

ANALYSIS

Schonheit et al. (1982), Kristjansson et al. (1982), and Lovley et al. (1980) showed that the acetoclastic SRB had a lower half-saturation constant, Ks, than acetoclastic methane producing bacteria (MPB). A mechanism was pro­posed whereby SRB could outcompete MPB at low substrate concentrations. Middleton and Lawrence (1977) implied that the entire carbon flow in a reactor receiving high substrate concentration (acetate) could be diverted to sulfur reduction. Similarly, an exclusively sulfate reducing system was dem­onstrated by Saw et al. (1987). On the other hand, Isa et al. (1986a, 1986b), Kroiss (personal communication, 1987), Szendry (1983), and McKinney (personal communication, 1986) have shown that methane production can proceed nearly uninhibited in the presence of 1,000 mg/L S6+ in the feed. The experience of Szendry (1983) was with distillery wastes, that of Kroiss (personal communication, 1987) was with citric acid plant effluent, that of Isa et al. (1986a, 1986b) with acetate and ethanol and that of McKinney (personal communication, 1986) with acetate and sodium sulfate in the lab­oratory. These experiences of uninhibited methanogenesis were observed in spite of very low doubling times of SRB as recently reported by Nanninga and Gottschalk (1987).

The reactors operated by Kroiss and Plahl-Wabnegg (1983) and Kroiss (personal communication, 1987) had total sulfide as high as 450 mg/L at pH 7.3. McKinney (personal communication, 1986) noted that sulfate re­duction was minimal at 1,000 mg/L TS and pH 8.4 and that the carbon flow was diverted towards methanogenesis. In all three cases, the un-ionized H2S was maintained well below the 200 mg/L level.

The self-inhibition of sulfate reducers by the buildup of product, dem­onstrated here, appears to be a mechanism whereby methanogens could out­compete sulfate reducing bacteria for carbon. Under conditions of high sul­fide concentrations and an elevated pH, the carbon flow can be diverted from sulfate reduction to methane production. This is in spite of the theoretical

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energy yields which favor the sulfide generation pathway. This may explain the apparent contradiction between the findings of Schonheit et al. (1982), Kristjansson et al. (1982), Middleton and Lawrence (1977), and Saw et al. (1987) as opposed to the findings of Isa et al. (1986a, 1986b), Kroiss (per­sonal communication, 1987), Cappenberg (1975), McKinney (personal com­munication, 1986), and Szendry (1983). In the former cases, sulfate reduc­tion accounted for the entire carbon flow. In .the latter cases, the carbon flow was primarily diverted towards methanogenesis.

ENGINEERING SIGNIFICANCE

The research presented here has several drawbacks. The most significant are inherent to batch studies, i.e., inability to differentiate between the pop­ulation shift, acclimation, and genuine metabolic rate retardation. Others are lack of gas stripping and the fact that HRT = SRT in serum bottles. Thus it is difficult if not impossible to infer conclusions as to the corresponding effects in full scale continuous flow reactors. Nevertheless, certain trends were indicated by this research. It appears that the engineer could choose the path of carbon utilization. On one hand, sulfate reduction could perhaps be optimized at the expense of methane production by minimization of total sulfide. Gas stripping of sulfate loaded anaerobic reactors has led to the creation of conditions favoring SRB in the treatment of industrial wastes and municipal sludge (Olthof et al., 1986). On the other hand, methane pro­duction could be optimized by maintenance of pH control and by allowing for an appreciable increase of the content of total sulfide. High rate fixed film reactors, upflow sludge bed reactors, and contact digesters offer a sig­nificantly elongated SRT required by MPB at high TS levels, to allow for manipulation of the carbon flow between the sulfide or methane producing bacterial systems.

SUMMARY AND CONCLUSIONS

Summary Toxicity of un-ionized H2S to the acidogenic and methanogenic processes

was manifested as a decreased rate of substrate utilization. Complete utili­zation of lactose and acetate substrates was achieved by increasing the re­tention time in batch cultures.

Experiments demonstrated that the quantity of sulfate reduced in a batch culture incubated with lactate was not dependent upon pH but was a function of the total sulfide (TS) concentration. Minimal sulfur reduction occurred when the total reactor (syringe) sulfide concentration was 1,000 mg/L TS. On the other hand, complete utilization of acetate and lactate was demon­strated in syringes with 1,000 mg/L TS (Figs. 2 and 3).

Sulfate reduction was found to be less a function of un-ionized H2S than total sulfides. The carbon flow could be diverted from sulfate reduction to methane production, as long as the H2S fraction was minimized to levels not inhibitory to MPB.

The literature has suggested that sulfate reducers would always outcom-pete methanogens for substrate. This research implied that when un-ionized H2S was maintained below 200 mg/L but TS were high (>400 mg/L), at high HRT, the carbon flow was diverted towards methane production, even

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when the preferred carbon source, lactate, was provided for the incompletely oxidizing sulfate reducing bacteria.

Conclusions 1. Lactose uptake and utilization rate increased as pH decreased without sul­

fide present. The uptake of lactose decreased in proportion to the concentration of un-ionized H2S.

2. Acetate utilization was inhibited by un-ionized H2S, more than total sul­fides.

3. Completion of lactose and acetate utilization processes under higher con­centrations of sulfides required a prolonged retention time.

4. Sulfate reduction was inhibited in proportion to the TS concentration, not the concentration of H2S.

5. In the presence of high TS and high pH (low concentrations of H2S), the carbon removal process was via the methanogenic pathways. At lower pH values (high H2S fraction), both the methanogenic and sulfate reducing pathways were inhibited.

ACKNOWLEDGMENTS

The research was sponsored by the Natural Sciences and Engineering Re­search Council (NSERC) of Canada through a strategic grant, and an op­erational grant to one of the writers (J.A.O.). Portions of this paper were presented at the 42nd Purdue University Industrial Waste Conference, May 1987. The writers acknowledge the constructive input from Dr. G. Blank, Dr. A. B . Sparling, and the excellent technical assistance from Mrs. J. Tin-gley.

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