APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1986, p. 580-5870099-2240/86/030580-08$02.00/0Copyright C 1986, American Society for Microbiology

Sulfate Reduction Relative to Methane Production in High-RateAnaerobic Digestion: Microbiological AspectsZAID ISA, STEPHANE GRUSENMEYER, AND WILLY VERSTRAETE*

Laboratory of Microbial Ecology, State University of Ghent, Coupure L 653, B-9000 Ghent, Belgium

Received 5 August 1985/Accepted 9 December 1985

In the high-rate anaerobic reactors studied (ca. 10 g of chemical oxygen demand [COD] removed per literof reactor per day), the sulfate-reducing bacteria (SRB) were poor competitors of methane-producing bacteria(MPB), scavenging only on the order of 10 to 20% of the total electron flow. The relatively noncompetitivenature of the SRB in this type of reactor is in sharp contrast to the tendency of the SRB to dominate in naturalenvironments and in other types of anaerobic digesters. Various factors such as the feedback inhibition of 112Son the SRB, iron limitation, the origin of the SRB inocula, biokinetics, and thermodynamics were investigated.The outcome of the SRB-MPB competition under the reactor conditions studied appeared to be particularlydetermined by two factors. The SRB, as predicted by the Vmaxm-K kinetics, competed most effectively at lowsubstrate levels (<0.5 g of COD per liter). The MPB, however, appeared to colonize and adhere much moreeffectively to the polyurethane carrier matrix present in the reactor, thus compensating for the apparent lowergrowth rates. Even if the reactor was initially allowed to be predominantly colonized by SRB, the MPB couldregain dominance.

Several mechanisms have been proposed to explain theinhibitory effect of sulfate on methanogenesis in naturalecosystems and in anaerobic digesters. The toxicity ofsulfide or free H2S produced by the microbial reduction ofsulfate is often suggested as a factor of primary importance(5, 12, 14, 21). The kinetics of competition for the availableelectron donors between sulfate-reducing bacteria (SRB)and methane-producing bacteria (MPB) have also receivedconsiderable attention (1, 11, 19). The SRB apparently havea higher affinity (lower Km) for hydrogen and acetate, whichare the major methane precursors, relative to the MPB(Table 1). This enables them to maintain the pool of thesesubstrates at concentrations too low for the MPB whensulfate is not limiting. The Km value for hydrogen is about0.002 mg/liter (0.001 mM) for the SRB and 0.012 mg/liter(0.006 mM) for the MPB. In the case of acetate as an electrondonor, the Km values for the SRB and MPB are, respec-tively, 12 mg/liter (0.2 mM) and 180 mg/liter (3 mM). Finally,from a purely thermodynamical point of view, the reductionof sulfate is energetically slightly more favorable than thereduction of bicarbonate (10).The SRB are normally dominant in natural ecosystems

such as freshwater and marine sediments and also inanaerobic digesters where methanogenesis was found to beinhibited by the presence of sulfate. The dominance of theSRB is indicated by the higher percentage of the substrateelectrons (in term of chemical oxygen demand [COD])partitioned by the SRB compared with those partitioned bythe MPB (Table 2). In freshwater and marine sediments,between 75 and 99% of the substrate electrons appear to bescavenged by the SRB. In anaerobic digesters, the SRB canutilize between 53 and 92% of the available substrate elec-trons.As reported earlier (9), the presence of high levels of

sulfate (up to 5 g of sulfate S per liter) did not significantlyinhibit methane production from synthetic media containing

* Corresponding author.

acetate (medium A) or acetate together with ethanol (me-dium AE) digested in high-rate anaerobic reactors. Thepurpose of this paper is to examine in more detail why theSRB were apparently not able to scavenge the major part ofthe electron flow under those conditions. Several factorswhich could affect the activities of the SRB were investi-gated. These include the possible feedback inhibition of H2Son SRB, iron availability for the SRB, the effect of the SRBinocula, substrate concentration, and the capacity of theSRB to colonize and adhere in the carrier matrix of thereactor. The effect of iron availability was investigatedbecause it has been reported that the SRB have an absoluterequirement for iron (2, 13, 17). A deficiency of this elementcould severely affect the growth of the SRB.

MATERIALS AND METHODS

Experimental set-up. Two high-rate anaerobic reactors withreticulated polyurethane sponges as a carrier matrix for themicroorganisms were used. The synthetic media digested inthese reactors contained acetate (medium A) or acetate plusethanol (medium AE). The details of the reactors and the

TABLE 1. Values of K,,, and Vmax for acetate and hydrogen ofSRB and MPB

Vmax (mMK,,, (mM Refer- substrate Refer-Bacteria Substrate subtr(mMef e

removed/g ef esubstrae) ence of VSS"~ecper day)

SRB Acetate 0.2 19 74 8

Hydrogen 0.001 11 112 18

MPB Acetate 3.0 19 45 23Hydrogen 0.006 11 123 18

VSS, Volatile suspended solids.

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TABLE 2. Partitioning of electron equivalents (in terms of COD) by SRB and MPB in natural environments and in anaerobic digesters

Equivalent COD (mg/liter) % Electron flowEnvironment SQ20 S CH4 produced 2- S Reference

reduced (mM) (MM) S04 CH4 SRB MPBreduced produced

Freshwater sediments 0.328a 0.00025a 20.99 0.016 99.92 0.08 260.859b 0.00012b 55.00 0.008 99.99 0.01 260.0589 0.0194 3.77 1.242 75.20 24.80 150.880 0.004 56.32 0.256 99.50 0.50 16

Marine sediments 50.00c 0.3c 3.20" 0.019d 99.40 0.60 209.84e 0.634e 62.98 f 4.06f 93.94 6.06 1

Anaerobic digesters 13.53 11.81 865.9 755.8 53.40 46.60 1416.66 1.39 1068.8 88.96 92.30 7.70 1429.06 2.68 1859.8 171.5 91.60 8.40 3

a 18 cm below sediment surface.b On sediment surface.c Nanomoles per gram of sediment per hour.d Milligrams per kilogram of sediment per hour.e X 10-4 mmol/kg of sediment.f Milligrams per kilogram of sediment.

composition ofthe media are similar to those described earlier(9).

In all experiments involving the high-rate anaerobic reac-

tors, the influents (media A and AE), containing 0.030 g ofsulfate S per liter, were supplemented with 10 g of sulfate Sper 100 g of removable COD (except where indicated) andwere digested in the reactors at the volumetric loading rateof 10 g of COD per liter of reactor per day. After 24 h ofdigestion, the mixed liquor in the feed reservoir was sampledfor analysis and replaced with new influent. Similarly, thebiogas produced was also sampled daily for analysis. Theexperiment was continued until a steady-state condition hadbeen reached. Details of each experiment are as follows.

Effect of hydrogen sulfide stripping. The biogas producedfrom the anaerobic digestion of medium AE supplementedwith 0.5 g of sulfate S per liter was continuously stripped intoa flask containing 100 ml of zinc acetate solution to remove

the H2S through precipitation as zinc sulfide. In a controlreactor, the biogas was stripped similarly but into a flaskcontaining 100 ml of distilled water.

Effect of iron availability. The iron concentration of me-

dium AE was varied as follows: 0 g/liter, 0.02 g/liter (normaliron level of the medium), 2.03 g/liter (excess iron level of themedium), and 0.02 g/liter but chelated with 2,2-dipyridyl.The concentration of 2,2-dipyridyl added was 100 mg/liter.

Effect of SRB inocula. The media were digested in twoseparate reactors. The first reactor was initially inoculatedwith 100 ml of well-digested anaerobic sludge from a similarreactor treating distillery wastewater rich in sulfate as a

source of freshwater SRB. The second reactor was startedup by inoculation with 10% (wt/vol) marine mud collected

from the sea 5 km off the Zeebrugge (Belgium) coast as a

source of marine SRB. In addition, the media were supple-mented with 12.5 g of NaCl per liter and 2.5 g of MgCl2 perliter to give half-strength seawater salinity.

Effect of substrate concentrations. Medium AE with dif-ferent COD levels, namely, 5.0, 1.0, and 0.5 g/liter, andsupplemented with 0.5 g Qf sulfate S per liter was digested insimilar reactors at a volumetric loading rate of 10 g of CODper liter of reactor per day.

Effect of hydraulic residence time (OH). Media A and AEsupplemented with 0.5 g of sulfate S per liter were digestedin high-rate reactors at a volumetric loading rate of 10 g ofCOD per liter of reactor per day and at two differenthydraulic residence times, namely 0.5 and 10 days.

Activities of biomass adhering to sponges and washed out ofthe reactor. Two batch-type anaerobic reactors were used.The first contained 500 ml of the effluent from the high-rateanaerobic reactor digested with medium AE and supple-mented with 0.5 g of sulfate S per liter. To this was added 500ml of medium AE to give a final COD concentration of 5g/liter. Finally, this mixed liquor (1 liter) was supplementedwith 1.0 g of sulfate S per liter. Three pieces of sponges froma well-operated high-rate anaerobic reactor which had beenfed with medium AE supplemented with sulfate were

squeezed into the second batch reactor containing 1 liter ofmedium AE (COD = 5 g/liter) and supplemented with 1.0 gof sulfate S per liter. The number of sponges were chosen insuch a way that it gave a similar level of volatile suspendedsolids as for the first batch reactor. The biogas produced, theamounts of sulfate reduced, and COD removed from the twobatch reactors were determined.

TABLE 3. Effect of constant H2S stripping on the competitivity of SRB and MPB (n = 3)

Biogas Composition of biogas Specific yield % Electron flow%SO2- S produced of methane

Reactore % 4 (litersliter of (ml of CH4/greduced reactor per % CH4 % CO2 % H2S of COD SRB MPB

day) removed)

A 65.3 + 2.5 3.31 ± 0.07 83.8 ± 1.1 14.8 ± 1.1 1.5 + 0.2 290 ± 17 14.6 ± 0.3 85.4 ± 0.3B 71.0 ± 6.0 3.23 ± 0.09 85.1 ± 1.9 14.9 + 1.9 0 264 ± 16 15.3 ± 1.3 84.7 ± 1.3

a Reactor A, Constant stripping of biogas into distilled water (control reactor); reactor B, constant stripping of biogas into zinc acetate solution. In all cases.loading rate = 10 g ofCOD per liter of reactor per day. Amount of sulfate added = 0.5 g of sulfate S per liter (or 10 g of sulfate S per 100 g of removable COD).

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TABLE 4. Effect of iron availability on the competitivity of SRB and MPB (n = 5)

Biogas Composition of biogas Specific % Electron flowron

2 produced yield ofadded % S04 S ltr/ltro % COD methane (ml

(g/liter)a reduced reactor per % CH4 % Co, % H2S removed of CHdg of SRB MPBday) ~~~~~~~~~~CODday) removed)

0 72.0 ± 2.0 3.17 ± 0.09 84.3 + 1.7 14.3 ± 1.8 1.43 ± 0.20 84.7 ± 3.0 302 ± 15 16.9 ± 0.9 83.1 ± 0.90.02b 65.0 ± 3.1 3.19 ± 0.40 82.4 ± 0.6 16.1 ± 0.5 1.51 ± 0.16 90.5 ± 8.0 298 ± 46 11.1 ± 2.6 88.9 ± 2.62.03 73.3 ± 1.2 3.13 ± 0.06 85.2 ± 1.6 14.3 ± 1.2 0.21 ± 0.09 92.8 ± 2.0 272 ± 2 16.0 ± 3.2 84.0 ± 3.20.02' 78.8 ± 5.2 2.45 ± 0.62 86.7 + 1.3 11.1 ± 1.3 2.20 ± 0.30 66.4 ± 13.3 290 ± 20 21.9 ± 0.8 78.1 ± 0.8

' In all cases, loading rate = 10 g of COD per liter of reactor per day. Amount of sulfate added = 0.5 g of sulfate S per liter (or 10 g of sulfate S per 100 g ofremovable COD).

Normal iron concentration of medium AE.'Normal iron concentration of medium AE but chelated with 100 mg of 2,2-dipyridyl per liter.

Effect of precolonization of the reactor with SRB. In a

separate experiment, a high-rate anaerobic reactor was firstprecolonized with predominantly SRB. Uncolonized reticu-lated polyurethane sponges were preinoculated with 100 mlof the SRB enrichment culture (SRB population, ca. 107/ml).The reactor was operated with medium AE supplementedwith 0.5 g of sulfate S per liter at a volumetric loading rate of10 g of COD per liter of reactor per day. The mixed liquor inthe feed reservoir was sampled for analysis and replacedwith new influent after 24 h of digestion. After ca. 10 days inoperation, the reactor was reinoculated with 100 ml ofwell-digested anaerobic liquor (rich in MPB) obtained from asimilar reactor treating sulfate-free wastewater. The reactorwas kept in operation under these conditions for a period of7 weeks.

Determination of the number of SRB. The number of SRBwas determined by plate count techniques with API medium(E. Merck AG, Darmstadt, Federal Republic of Germany).The composition of this medium was (grams/liter): sodiumlactate, 4.0; yeast extract, 1.0; ascorbic acid, 0.1; magne-sium sulfate, 0.2; dipotassium hydrogen phosphate, 0.01;ferrous ammonium sulfate, 0.2; sodium chloride, 12.0;resazurin-sodium, 0.001; and agar, 12.0.

Determination of the number of MPB. The number of MPBwas estimated by direct epifluorescence microscopy with a

Burker counting chamber and a Polyvar microscope. In thismethod the number of MPB was determined based on thespecific characteristic of the MPB to fluoresce under UVlight at 420 nm.

Percent electron flow by SRB and MPB. In the anaerobicdigestion of media rich in sulfate, the substrate electrons (interms of COD) are normally partitioned between the SRBand MPB. The electron flow by the SRB and MPB can becalculated from the following equations:

(a) By the SRB

Sulfate reduction: 4H2 + H+ + S042 -> HS- + 4H20 (1)

(2)CH3COO- + S42 -> HS- + 2HC03-

The COD of the H2S produced is given by:

H2S + 202 -- H2SO4Thus, 1 mol of sulfate reduced - 1 mol of H2S produced - 2mol of COD - 64 g of COD.Electron flow by the SRB = moles of sulfate S reduced x 64g = A g.

(b) By the MPB

Methane production: 4 H2 + HC03- + H+ --

CH4 + 3H20

CH3COO- + H20 -O CH4 + HC03-The COD of the CH4 produced is given by:

CH4 + 202 -- CO2 + 2H2O

(4)

(5)

(6)

Thus, 1 mol of CH4 produced 2 mol of COD _ 64 g of COD.Electron flow by the MPB = moles of CH4 produced x 64 g= B g.Therefore:

Percent electron flow by SRB = [A/(A + B)] x 100

Percent electron flow by MPB = [BI(A + B)] x 100

Free energy from sulfate reduction and methane produc-tion. The free energy evolved during the microbial reductionof sulfate and during methane production from acetate canbe calculated from the following equations (22):

TABLE 5. Effect of the SRB inocula on the competitivity of the SRB and MPB (n = 5)

Biogas Composition of biogas Specific yield % Electron flowOrigin %S42 prdcdof methaneMedium of SRB %educed S liproducted (ml of CH4/g

inoculaf" reduc (liters/liter % CH4 % CO, % H,S of COD SRB MPBof reactor remvedper day) removed)

A F 30.8 ± 6.0 3.0 ± 0.1 91.8 ± 1.2 8.0 ± 1.2 0.29 ± 0.04 297 ± 7 8.0 ± 0.3 92.0 + 0.3M 25.8 ± 4.4 3.6 ± 0.4 88.0 ± 2.3 11.9 ± 2.3 0.06 ± 0.02 320 ± 20 6.0 ± 0.7 94.0 + 0.7

AE F 65.0 ± 3.1 3.5 ± 0.3 82.7 ± 0.6 15.9 ± 0.5 1.46 ± 0.13 298 ± 50 11.1 ± 2.6 88.9 + 2.6M 75.1 ± 1.1 3.3 ± 0.1 89.6 ± 1.9 9.4 ± 2.0 1.02 ± 0.50 313 ± 7 16.6 ± 0.5 83.4 ± 0.5

F, Freshwater; M, marine. In all cases, loading rate = 10 g of COD per liter of reactor per day. Amount of sulfate added = 0.5 g of sulfate S per liter (or10 g of sulfate S per 100 g of removable COD).

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TABLE 6. Effect of substrate concentration on the competitivity of the SRB and MPB (n = 5)

Biogas Composition of biogas Specific yield C/Electron flow

InfluentC S042- S produced of methaneCOD reduced (liters/liter of (ml of CH4/g(g/liter)" reactor per % CH4 W. Co2 '/c H,S of COD SRB MPB

day) removed)

5.0 65.0 ± 3.1 3.19 + 0.40 82.4 + 0.6 16.1 ± 0.5 1.51 ± 0.16 298 ± 46 11.1 ± 2.6 88.9 ± 2.61.0 68.0 ± 1.5 2.02 ± 0.40 83.8 t 5.1 15.6 ± 5.1 0.60 ± 0.20 248 ± 56 27.4 ± 9.0 72.6 ± 9.00.5 43.5 ± 4.2 1.42 ± 0.26 72.8 + 2.1 26.8 ± 2.1 0.41 + 0.12 189 ± 73 34.2 + 6.3 65.8 ± 6.3

" In all cases, loading rate = 10 g of COD per liter of reactor per day. Amount of sulfate added = 10 g of sulfate S per 100 g of removable COD.

Sulfate reduction: CH3COO- + SO4--_ HS- + 2 HC03-AG"' = -47 kJ per reaction (7)

Methane production: CH3COO- + H2O = CH4 + HC03-AG0' = -31 kJ per reaction (8)

The AG0' is the increment of free energy for the reactorunder standard conditions (temperature, 273 K; pressure, 1atm [101.29 kPaJ), neutral pH, and products at 1 M (22). Thefree energy at pH 7 but at nonstandard concentration is givenby the following equations:

Sulfate reduction:

AG' -47 5.9 log [H3SHCO3]- kJ/reaction

[CH

(9)

Methane production:AG' = -31 + 5.9 log [HC03_ [CH4] kJ/reaction (10)

[CH3COO-

RESULTSFrom the results summarized in Table 3, it appears that

the stripping of H2S from biogas had no effect on the rate ofbiogas production and the percent electron flow by the SRB.The H,S stripping, however, increased the percentage ofsulfate reduced and lowered the specific yield of methane.Owing to the variability of the sampling and the analysis, thelatter trends, although evident, were not statistically signif-icant.

Table 4 summarizes the effect of iron availability for theSRB on sulfate reduction in medium AE digested in similarreactors. The results indicate that, at a sulfate level of 0.5 gof sulfate S per liter (or a ratio of 10 g of sulfate S/100 g ofremovable COD), the absence of added iron in medium AEresulted in similar volumes of biogas produced and compa-rable values in the percent electron flow by the SRB. Thepresence of normal (0.02 g/liter) and excess (2.03 g/liter)levels of iron did not appear to strongly influence the

competitivity of the SRB toward the MPB. The addition ofexcess iron gave a slightly lower specific yield of methaneand a better percent COD removal. The addition of 0.02 g ofiron per liter chelated with 2,2-dipyridyl decreased thevolume of biogas produced and the percent COD removal.On the other hand, the percent electron flow by the SRB andthe percentage of sulfate reduced increased by the additionof the same chelating agent.The results in Table 5 show that the marine SRB could not

compete better with the MPB than the freshwater SRBcould. This is indicated by the comparable values of thevolumes of biogas produced and also by the percent electronflow by the SRB from the reactors inoculated with thefreshwater SRB inocula and the marine SRB inocula, respec-tively.With regard to the effect of substrate concentration on the

competitive nature of the SRB relative to the MPB (Table 6),it was found that there was a substantial increase in thepercent electron flow by the SRB from 11 to 34% andconcomitantly a substantial decrease in the specific yield ofmethane from 298 to 189 ml of CH4 per g of COD removed,when the influent COD was decreased from 5 to 0.5 g/liter.

It was also noticed that the increase in hydraulic residencetime (OH) from 0.5 to 10 days (Table 7) increased the percentelectron flow by the SRB and also the percentage of sulfatereduced.The results in Table 8 reveal that the percent electron flow

by the SRB of the biomass from the sponges was much lower(26%) compared with that from the effluent (66%). Concom-itantly, the specific yield of methane was higher for thebiomass from the sponges (284 ml of CH4 per g of CODremoved) than that from the effluent (201 ml of CH4 per g ofCOD removed). The percent sulfate reduction was, respec-tively, 64 and 66% for the biomass from the sponges andfrom the effluent. The numbers of SRB and MPB present inthe sponges and in the effluent of the high-rate anaerobicreactor are given in Table 9. The number of SRB present inthe sponges was only about 30 times higher than the numberwashed out with the effluent. However, for the MPB, the

TABLE 7. Effect of hydraulic residence time (OH) on the competitivity of the SRB and MPB (n = 4)"

Biogas Composition of biogas Specific yield % Electron flowMedium O,, % SO42- S produced of methane

(days) reduced (liters/liter of% CH4 % CO, % H.S (ml of CH4/g SRB MPB

reactor per %C4 C C2/ HSofCODSRMPday ) removed)

A 0.5 27.0 ± 8.5 2.73 + 0.12 88.0 ± 1.4 11.9 + 1.5 0.18 ± 0.04 243 ± 9 6.4 ± 0.8 93.6 ± 0.810.0 34.6 + 7.7 2.99 ± 0.35 68.7 ± 4.0 30.3 ± 4.0 0.99 ± 0.14 213 ± 46 11.6 ± 1.3 88.4 ± 1.3

AE 0.5 65.0 + 3.1 3.19 0.40 82.4 0.6 16.1 0.5 1.51 + 0.16 298 46 11.1 2.6 88.9 2.6

10.0 98.4 + 2.6 3.35 ± 0.23 72.9 ± 3.0 25.1 ± 3.0 2.71 ± 0.30 267 + 41 16.7 ± 0.5 83.4 ± 0.5

"In all cases, loading rate = 10 g of COD per liter of reactor per day. Amount of sulfate added = 10 g of sulfate S per 100 g of removable COD.

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TABLE 8. Activities of biomass adhering to the sponges and biomass washed out in the effluent of a high-rate anaerobic reactor(11 = 4)'

2-Biogas Composition of biogas Specific yield of X, Electr-on flowSource of S04' S produced methane (ml ofbiomass reduced (liters)" % CH4 C/ CO. c H.S CH4/g of COD SRB MPB

removed)

Sponges 64.0 + 6.0 1.54 ± 0.35 82.0 ± 2.0 17.0 ± 2.6 1.1 ± 0.6 284 ± 30 26.0 ± 3.6 74.0 ± 3.6Effluent 66.0 ± 6.0 0.55 ± 0.24 47.3 ± 9.2 51.1 + 9.4 1.6 ± 0.6 201 + 48 66.2 ± 1.7 33.8 + 1.7

Amount of sulfate added = 1.0 g of sulfate S per liter.Total volume after 2 weeks of digestion.

number in the sponges was about 2,000 times higher than thenumber in the effluent.The results of the effect of precolonization of the reactor

with an SRB enrichment culture (Fig. 1; Table 10) show thatduring the first 10 days of digestion, the biogas productionwas very low and a high sulfate-reducing activity wasattained (percent sulfate reduction averaged 71.5% or about786 mg of sulfate S reduced per liter of reactor per day).During this period, the SRB outcompeted the MPB as shownby the higher percent electron flow by SRB (91%) and thelower specific yield of methane (135 mg of CH4 per g of CODremoved).

It was noticed that after the reactor was reinoculated withwell-digested anaerobic liquor, the biogas production in-creased slowly until it stabilized at an average of 3.6liters/liter of reactor per day after about 7 weeks. On theother hand, the percent sulfate reduction decreased from71.5 to 63.7 and 49.0%, respectively, after 2 and 7 weeks ofreinoculation. Concomitantly, the SRB were outcompetedby the MPB as indicated by a drop in the percent electronflow from 91.0 to 35.0 and 5.3%, respectively. These resultssuggest that the MPB were able to outcompete the SRB inthis type of reactor even though the reactor was initiallypredominated by the SRB.

DISCUSSIONButlin et al. (3) reported that the H2S produced from

microbial reduction of sulfate is inhibitory to the SRB. Aslight inhibition was shown in a previous study (9). Theconstant H.S stripping experiment confirms that the SRB areonly very slightly affected by the H2S produced from thereduction of sulfate. It can be concluded that the lack of aninhibitory effect of sulfate on methane production as found inour studies was not due to feedback inhibition by H.S on theSRB.

Callander and Barford (4) have shown with regard to ironavailability that in anaerobic digesters rich in sulfide, metalnutrients are mainly precipitated as insoluble sulfides. Yet.the authors stressed the fact that the formation of solubleorganic metal complexes determines the availability of sol-uble metals in the digesters. Hoban and Van den Berg (7) andCallander (I. J. Callander, Ph.D. thesis, University of

TABLE 9. Estimation of the SRB aind MPB from sponges andeffluent of a high-rate anaerobic reactor upon digestion with

medium AE

SaMPIC SR~5B MB(lm Ratio ofSample tnl/mI) MPB ()7/mI) MPB/SRBSponges 12 xI.. 4.8 x 10'' 10'Effluent 4 x 104 2.6 x 1() 103

Ratio of sponges/effluent 30 2,000

Sydney, Sydney, Australia, 1982) observed the beneficialeffect of iron supplementation on methane production. Inour experiments, the omission or extra addition of iron didnot appear to have a strong influence on the methane andsulfide formation processes (Table 4). The addition of 2,2-dipyridyl as a specific chelator for iron appeared to inhibitthe MPB, however. From these results, it appears that theabsence of the inhibitory effect of sulfate on methane pro-duction in our study was not due to iron limitation for theSRB. The iron was made available to the SRB in thissulfide-rich environment probably by chelation with organicligands present in the yeast extract added to the media.When acetate was used as a source of reducing equiva-

lents, marine SRB were not better competitors of the MPBrelative to freshwater SRB (Table 5). Acetate is possibly nota suitable electron donor for the marine SRB, althoughWiddel and Pfennig (25) reported that one species of the SRB(Desulfobacter postgatei) which is present in brackish waterand marine sediments uses acetate as the only electrondonor. When acetate and ethanol were used as a source ofreducing equivalents, the marine SRB were slightly bettercompetitors for the MPB as indicated by a slight increase inthe percent electron flow by the SRB and the percentage ofsulfate reduced. Yet they could not outcompete the MPB.Thus, the relatively lower levels of sulfate reduction ob-served in our reactor systems are probably not due to lack ofan appropriate inoculum.The competitive nature of the SRB relative to the MPB

was markedly increased by decreasing the substrate concen-tration (Table 6). The fact that at lower substrate concentra-

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4

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100

80

40

n

FIG. 1. Effect of precolonization of the SRB followed byreinoculation with anaerobic liquor rich in MPB on the competitivityof the SRB and MPB. A. Rate of biogas production, B, percentageof methane in biogas.

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TABLE 10. Effect of precolonization of the SRB in the reactor followed by reinoculation with anaerobic liquor rich in MPB on thecompetitivity of the SRB and MPB (n = 7)0

Sulfate Composition of biogas % Electron flowreducing Biogas Specific

P%2- S activity (mg producedyield of

Period % S04- Of S042 5 rdue methane (ml(days) reduced reduced/ (fiters/liter % CH4 % C02 % H2S of CH4/Of SRB MPB

liter of of reactor CODreactor per per day) removed)

day)

1-10 71.5 ± 3.2 786 ± 50 0.2 ± 0.1 26.8 ± 9.1 64.9 ± 12.0 8.3 ± 8.0 135 ± 105 91.0 ± 6.5 9.0 ± 6.511-24 63.7 ± 11.2 704 ± 127 0.9 ± 0.4 61.6 ± 16.1 35.7 ± 15.2 2.5 ± 1.1 1% ± 70 35.0 ± 9.7 65.0 ± 9.754-63 49.0 ± 5.0 521 ± 72 3.6 ± 0.3 86.3 ± 2.9 12.4 + 3.1 1.4 ± 0.1 320 ± 9 5.3 ± 0.7 94.7 ± 0.7

a The reactor was preinoculated with an SRB enrichment culture at day 0 and reinoculated with well-digested anaerobic liquor (rich in MPB) at day 11. In allcases, loading rate = 10 g of COD per liter of reactor per day. Amount of sulfate added = 0.5 g of sulfate S per liter (or 10 g of sulfate S per 100 g of removableCOD).

TABLE 11. Partitioning of the substrate electrons by SRB and MPB from various samples

Sample S042- S COD g of S042- % Electron flow Effect on methane Reference(g/liter) (g/liter) S/100 g of COD SRB MPB productiona

Distillery wastewater 0.20 4.50 4 26.1 73.9 Slight inhibition 6Pulp industry wastewater 0.77 11.50 7 0.4 99.6 No inhibition 24Citric acid factory wastewater 0.58 8.92 7 16.7 83.3 Slight inhibition 12Citric acid factory wastewater 1.27 10.50 12 27.3 72.7 Slight inhibition 12Sewage sludge 1.20 1.80 67 53.4 46.6 Strong inhibition (58) 14Sewage sludge 1.60 1.80 89 92.3 7.7 Strong inhibition (78) 14Sewage sludge 11.76 2.75 428 91.6 8.4 Strong inhibition (91) 3

a Figures in parentheses indicate the percent inhibition of methane production.

tions the SRB tend to be more competitive can be backed upby some data from the literature (Table 11). Yet, in our studyat the lowest substrate concentration investigated (i.e., 0.5 gof COD per liter per day) and at a ratio of 10 g of sulfateS/100 g of removable COD, the SRB were still largelyoutcompeted by the MPB. At this substrate concentration,the percent electron flow by the SRB and MPB is 34.2 and65.8%, respectively. The percent electron flow by the SRBand MPB was also calculated from the data reported else-where (9). From Table 12, it appears that increasing thesulfate concentration or sulfate/COD ratio increased the

TABLE 12. Partitioning of the substrate electrons by SRB andMPB from synthetic media upon digestion in high-rate anaerobic

reactors (n = 5)YSO42- S added % Electron flow

Medium gg/liter COD SRB MPB

A 0.1 2 2.7 0.3 97.3 0.30.2 4 3.6 0.9 96.4 0.90.3 6 5.9 0.3 94.1 0.30.4 8 7.1 ± 0.4 92.9 ± 0.40.5 10 6.8 ± 0.8 93.2 + 0.85.0 100 6.8 ± 1.9 93.2 ± 1.9

10.0 200 34.3 ± 3.7 65.7 ± 3.7

AE 0.1 2 3.4 ± 0.2 96.6 ± 0.20.2 4 6.7 ± 0.2 93.3 ± 0.20.3 6 8.8 ± 0.7 91.2 ± 0.70.4 8 8.3 ± 0.5 91.7 ± 0.50.5 10 11.3 ± 0.8 88.7 ± 0.85.0 100 18.5 ± 0.8 81.5 ± 0.8

10.0 200 33.4 ± 0.8 66.6 ± 0.8

a Volumetric loading rate = 10 g of COD per liter of reactor per day.

percent electron flow by the SRB. The percent electron flowscavenged by the SRB was generally higher in medium AEthan in medium A. This again indicates that acetate alone isnot a suitable substrate for the SRB, possibly because ityields a much lower free energy (47 kJ per reaction) com-pared with ethanol (195 kJ per reaction) (22).On the basis of the Vma and Km values given in Table 1,

the curves representing the rate of substrate removal as afunction of substrate concentration were plotted for acetate(Fig. 2) and hydrogen (Fig. 3). It appears that, for acetate,the SRB theoretically always outcompete the MPB. Yet, theratio of growth rates of the SRB relative to the MPB isparticularly high at low levels of acetate (1 to 5 mM). Our

11

0

FIG.VSRB/VM

Ratio V SRB/V MPB

Acetate (mM)0

1 2 3 4 5 6 7 8 9 102. Acetate removal rate of SRB and MPB and the ratio of[PB-

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0 . 01 . 02 . 03 . 04 . 05 . 06 . 07 . 08 . 09 . 1FIG. 3. Hydrogen removal rate of the SRB and MPB and the

ratio of VSRB/VMPB-

data indicate that at a low substrate level (16 mM in theinfluent, 8 mM in the effluent), the SRB indeed increase theirshare of the electron flow. For hydrogen, the SRB onlyappear competitive with the MPB at levels of hydrogenbelow 50 ,uM (Fig. 3). The concentrations of hydrogenobserved for medium AE were on the order of 0.002 to0.07% in the gas phase or 0.04 to 1.4 ,uM in solution. Hencethe major amount of H2 flow should be scavenged by theSRB. As indicated previously (9), apparently 50% of theavailable H2 is captured by the SRB. Hence, an ecologicalparameter other than the Vmax-Km combination must beinfluencing the SRB-MPB competition.According to equations 7 and 8, under standard condi-

tions, 1 mol of acetate used to reduce sulfate yields 47 kJcompared with only 31 kJ for the production of methane.Using equations 9 and 10, the change in free energy wascalculated for the concentrations of acetate and sulfate in thetest runs. For instance, at a sulfate level of 0.5 g of sulfate Sper liter (10 g of sulfate S per 100 g of removable COD), theaverage concentrations of HCO3f, HS-, and CH3COO-were, respectively, 117.3, 2.9, and 5.3 mM. The potentialfree energy therefore becomes 49 kJ. The AG' for theacetogenic MPB under similar conditions amounts to 32 kJ.It thus appears that, thermodynamically, the use of acetateas an electron donor for sulfate reduction is more favoredthan for the production of methane. Hence, the dominanceof the MPB in the present study cannot be explained fromthe thermodynamical point of view.The increases in the percent electron flow by the SRB and

the percentage of sulfate reduction as a result of the increasein hydraulic residence time (OH) from 0.5 to 10 days (Table 7)both indicate that the wash-out of the SRB from the reactorcould be at the basis of the restricted competitive nature ofthe SRB in the present study. In other words, it could be thatthe SRB colonize the carrier matrix poorly compared withthe MPB. This hypothesis is supported by the results shownin Tables 8 and 9. The electron flow by the biomass squeezedfrom the sponges was much more directed toward methaneproduction than was that of the biomass washed out of thereactor. Concomitantly, the biomass from the sponges re-sulted in a higher specific yield of methane (284 ml of CH4per g of COD removed) than the washed-out biomass (201 mlof CH4 per g of COD removed). Table 9 further indicates thatmore SRB are washed out from the reactor compared with

MPB. The ratio of the SRB present in the sponges to thosein the effluent was only about 30, whereas for the MPB, theratio was about 2,000. This indicates a better colonizationand adherence of the MPB in the carrier matrix (sponges)than the SRB, which enables them to outcompete the SRB,even at higher sulfate levels. Hence, the capacity to colonizeand adhere to the carrier matrix inside the reactor appears tobe of major importance with respect to the competitionbetween SRB and MPB. It is not clear what characteristicsof the MPB cells and of the carrier matrix in particulardetermine this apparently selective colonization.

In conclusion, the inability of the SRB to outcompete theMPB in the present study can be attributed to the highsubstrate concentration on the one hand and the preferentialcolonization of the carrier matrix by the MPB on the otherhand.

ACKNOWLEDGMENTSThis work was financially supported by the Algemeen Bestuur voor

Ontwikkelingssamenwerking (ABOS) of Belgium.We thank C. Graveel for her diligent typing of the manuscript.

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