APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1982, p. 1373-13790099-2240/82/061373-07$02.00/0

Vol. 43, No. 6

Kinetic Analysis of Competition Between Sulfate Reducersand Methanogens for Hydrogen in Sedimentst

DEREK R. LOVLEY,* DARYL F. DWYER, AND MICHAEL J. KLUG

Kellogg Biological Station, Michigan State University, Hickory Corners, Michigan 49060, and Department ofMicrobiology and Public Health, Michigan State University, East Lansing, Michigan 48824

Received 17 December 1981/Accepted 2 March 1982

The competition between sulfate-reducing and methanogenic bacteria forhydrogen was investigated in eutrophic lake sediments that contained low in situsulfate concentrations and in sulfate-amended sediments. Sulfate reduction andmethane production coexisted in situ in lake surface sediments (0 to 2 cm), butmethane production was the dominant terminal process. Addition of 10 to 20 mMsulfate to sediments resulted in a decrease in the hydrogen partial pressure and a

concomitant inhibition of methane production over time. Molybdate inhibition ofsulfate reduction in sulfate-amended sediments was followed by an increase in thehydrogen partial pressure and the methane production rate to values comparableto those in sediments not amended with sulfate. The sulfate reducer populationhad a half-saturation constant for hydrogen uptake of 141 pascals versus 597pascals for the methanogen population. Thus, when sulfate was not limiting, thelower half-saturation constant of sulfate reducers enabled them to inhibit methaneproduction by lowering the hydrogen partial pressure below levels that methano-gens could effectively utilize. However, methanogens coexisted with sulfatereducers in the presence of sulfate, and the outcome of competition at any timewas a function of the rate of hydrogen production, the relative population sizes,and sulfate availability.

It is generally considered that sulfate-reducingbacteria (SRB) can inhibit the activity of meth-anogenic bacteria (MB) when millimolar quanti-ties of sulfate are present. Thermodynamic cal-culations can be used to predict the exclusion ofmethane production in sulfate-containing sedi-ments (8, 14, 29). However, it is invalid to arguethat a reaction that is more thermodynamicallyfavorable will exclude another reaction that isalso thermodynamically favorable (15). There-fore, MB must be inhibited by toxic metabolites,the lack of methane precursors, or requiredgrowth factors in the presence of sulfate. Theprevalent conclusion is that SRB inhibit MB byoutcompeting them for hydrogen and acetate (1,2, 6, 14, 18, 21, 29), but the mechanism(s) forthis have not been elucidated. MB are frequentlypresent in sulfate-containing sediments and havethe potential to consume methane precursors asevidenced by methane production when sulfatereduction is inhibited or when hydrogen or ace-tate is added to the sediments (2, 21, 26, 29). Ourworking hypothesis was that SRB have a higheraffinity for hydrogen and acetate than MB,which enables SRB to maintain the pool of thesesubstrates at concentrations too low for MB to

t Articles no. 10275 of the Michigan Agricultural Experi-ment Station and no. 462 of the Kellogg Biological Station.

effectively utilize when sulfate is not limiting toSRB. The studies reported here concentrated onthe competition for hydrogen since acetate-uti-lizing MB are generally absent in natural sedi-ments in which SRB effectively outcompete MB(8, 19, 26), and the ultimate competition is thusfor hydrogen.

MATERIALS AND METHODS

Measurements of in situ rates. Sediments were col-lected during summer stratification from two sites inWintergreen Lake, a eutrophic lake located in south-western Michigan. During summer stratification thesediments at the profundal site, site A, lie below ananaerobic, sulfate-depleted hypolimnion (sulfate con-centration range, 30 to 160 FM), whereas those at thedepth of the thermocline, site B, have oxygen (range, 1to 4 mg of oxygen per liter) and sulfate (180 FM) in theoverlying water (16, 17).

Sulfate reduction rates were measured by the directinjection method of J0rgenson (10) as described indetail by King and Klug (11). Briefly, 10 ,ul of carrier-free 35So42- (1 ,uCi) was injected into sediment coresincubated at in situ temperatures. The incubation wasstopped by quick freezing. The 35S2- produced wasdistilled, trapped, and quantified by liquid scintillationcounting. Sulfate reduction rates were calculated bymultiplying the rate of conversion of 35S042- to 35s2-by the in situ sulfate pool. Interstitial water wascollected with dialysis samplers (17) and analyzed forsulfate turbidimetrically (28).

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Methane production was measured on 5-ml sub-cores taken through ports in cores (7-cm inner diame-ter) collected using SCUBA (self-contained underwa-ter breathing apparatus). The subcores were extrudedinto pressure tubes (Bellco Glass) or 20-ml serumbottles (Wheaton Scientific) under an atmosphere of93% nitrogen and 7% carbon dioxide. The vesselswere stoppered with butyl rubber stoppers (BellcoGlass), sealed with an aluminum crimp, and incubatedat in situ temperatures. The rate of increase in meth-ane concentration in the headspace was measured atintervals over a 20- to 30-h incubation period. Thetubes were shaken before each methane analysis toequilibrate the dissolved gases with the headspace.Methane was analyzed on a Varian 600D gas chro-matograph as described below.Laboratory studies. Sediments for laboratory studies

were collected from the A and B site with an Eckmandredge. Depending on the experiment 500, 700, or 800ml of sediment was transferred under anaerobic condi-tions to 1-liter reagent bottles (Wheaton Scientific) andsealed with a rubber stopper.A final concentration of either 10 or 20 mM ferrous

sulfate (sulfate-amended sediments) or ferrous chlo-ride (control sediments) was added to the sediments.Ferrous salts were used to prevent the accumulation offree sulfide, which is toxic to methanogens at highconcentrations (7, 29). Ferrous chloride was added tocontrol flasks to eliminate any potential differentialeffects of excess iron on hydrogen uptake or produc-tion. The sediments were incubated at 20 ± 2°C in thedark without mixing or were placed on a cell produc-tion bottle roller (Bellco) and slowly turned. Molyb-date was added to the sediments as a nitrogen-flushed0.5 M solution of sodium molybdate to give a finalconcentration of 5 mM. Molybdate is regarded as aneffective and specific inhibitor of sulfate reduction insediments (20, 21, 25, 26).Carbon dioxide and methane in the headspace of the

bottles were analyzed on a Carle basic gas chromato-graph equipped with a microthermistor detector. Thegases were separated on a 1-m column of Poropak N(Waters Associates) with a helium carrier at a flow rateof 20 ml/min and an oven temperature of 60°C. Whengreater sensitivity for methane was desired, a Varian600D gas chromatograph with a flame ionization detec-tor was used. Gases were separated with a heliumcarrier on a 1-m column of Poropak N at 50°C.Hydrogen was analyzed on a Varian 3700 gas chro-matograph with a thermal conductivity detector. Thegases were separated on a 3-m column of Poropak Nwith nitrogen as the carrier at 15 ml/min and an oventemperature of 35°C. The detection limit was 0.04pascals. One pascal is approximately equivalent to 9.9x 10-6 atm and a dissolved hydrogen concentration of8 nM. The bottles were shaken vigorously beforesampling to equilibrate the dissolved gases with theheadspace.

Interstitial water for sulfate analysis was collectedby centrifugation and analyzed by high-pressure liquidchromatography. Ions were separated at room tem-perature on a Vydac column (Anspec; 5 x 0.46 cm)with a solvent of 1 mM phthalic acid (pH 5.5) at a flowrate of 2 ml/min. Sulfate was detected with a Wescanconductivity detector (Anspec).For the kinetic analysis of hydrogen uptake, 4- or 6-

ml samples of sediments were dispensed into roll tubes

(25 x 142 mm; Bellco). The tubes were flushed withoxygen-free nitrogen before and during the transfer. Inexperiments where chloroform was added to sedi-ments, a 50- to 75-ml sample of sediment was firsttransferred to a 120-ml serum bottle. Chloroform wasadded directly (final concentration, 0.003% [vol/vol]).The sediments were mixed and dispensed into tubes asabove. The tubes were incubated with slow rolling ona tube roller to create a thin film of sediment (27).Hydrogen was added, and headspace samples werewithdrawn over time and analyzed for hydrogen ormethane or both.Two experimental approaches were used to ensure

that chloroform did not alter the potential of SRB totake up hydrogen. In the first experiment, sedimentswere amended with 550 ,uM (final concentration) sul-fate to saturate SRB for sulfate. The sediments wereincubated under saturating hydrogen (50 kPa) on thetube roller, and the rates of sulfate depletion over a 2-hincubation period in sediments treated with chloro-form and control sediments were compared. In thesecond experiment, sediments that had been adaptedto 20 mM sulfate were incubated on the tube rollerwith an initial hydrogen concentration of 1 kPa. Theinitial rate of hydrogen uptake was measured in un-treated sediments, sediments treated with chloroform,and sediments treated with molybdate. If chloroformdid not inhibit hydrogen uptake by SRB, then the sumof hydrogen uptake in sediments treated with chloro-form and sediments treated with molybdate wouldequal the hydrogen uptake in untreated sediments.

Kinetic analysis. Hydrogen uptake in sediments haspreviously been shown to follow Michaelis-Mentenkinetics (27).

V VM X SK+ S (1)

where V is the velocity of uptake, VM is the maximumpotential uptake velocity, S is the substrate concentra-tion, and K is the substrate concentration at which V =0.5 VM. Kinetic parameters were estimated from prog-ress curves of hydrogen consumption over time. Alinearized expression of an integrated form of theMichaelis-Menten expression can be derived (23).

In SSt = -1 X So - St + VMt K t K (2)

where So is the initial substrate concentration and S, isthe substrate concentration at time t. This methodgives kinetic parameters for hydrogen uptake in sedi-ments comparable to those estimated from initial ve-locity studies (27) and has the added advantage thatvariability between sediment samples for a particularkinetic analysis can be eliminated since all the sub-strate concentrations are, in effect, tested on the samesediment sample.Sediments containing hydrogen-consuming MB and

SRB populations can be expected to have a totalhydrogen uptake described by a two-term Michaelis-Menten equation.

VTVMSRB X VMMB X(3)

KSRB + S KMB + S

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COMPETITION FOR HYDROGEN IN SEDIMENTS 1375

TABLE 1. Relative importance of methane production and sulfate reduction in the surface sediments (0 to 2cm) of Wintergreen Lake during summer stratification

Sediment Sulfate concn Methanea Sulfatea Sulfate reductionbsite (A.M) production reduction (% of total)

Ac 71 40 + 10 6.2 ± 1.7 13B 59 26±12 4.0±1.3 13

a Micromoles per liter of sediment per hour; mean ± standard error of 3 or more rate measurements.b Sulfate reduction rate divided by total of sulfate reduction rate and methane production rate.c Sulfate concentration and reduction rate for A site from King and Klug (11).

where VT is the total rate of hydrogen uptake, VMSRBand KSRB are the VM and K of the SRB population, andVMMB and KMB are the VM and K for the MB. Thistwo-term equation was used in the analysis of hydro-gen uptake in sulfate-containing sediments that hadboth MB and SRB populations. Kinetic parameters forthe two populations were entered into a programwhich calculated total hydrogen uptake over time.

RESULTS

Concurrent methane production and sulfatereduction were observed in the surface sedi-ments (0 to 2 cm) of both site A and site B (Table1). Methane production was the dominant proc-

ess and comprised about the same proportion ofthe total of methane production and sulfatereduction at both sites.Methane production in sediments from both

sites was completely inhibited within 2 to 5 daysat 20°C by the addition of 10 or 20 mM sulfate.Active sulfate reduction in the sulfate-amendedsediments was evidenced by the loss of dis-solved sulfate and the appearance of black fer-rous sulfide over time. There was also an in-crease in carbon dioxide production in sulfate-amended sediments over that in controlsediments.

Sulfate-amended sediments in which methaneproduction was inhibited had significantly lowerhydrogen partial pressures than FeCl2 controlsand untreated sediments (Table 2). Monitoringover time demonstrated that the inhibition ofmethane production and the decrease in hydro-gen were concurrent (Fig. 1). Both control andsulfate-amended sediments had high initial ratesof methane production and elevated hydrogenpartial pressures, presumably due to distur-bances in carbon flow resulting from the initialmanipulations with the sediment. The hydrogenpartial pressure stabilized in control (FeCl2-amended) sediments at approximately 1 Pa,whereas methane production continued at lowerrates. However, in sulfate-amended sedimentsthe methane production rate and hydrogen par-tial pressure dropped sharply until methane pro-duction was no longer detectable. The hydrogenpartial pressure continued to slowly decline aftermethane production had ceased.

TABLE 2. Hydrogen partial pressure in sedimentswith and without added sulfate

HydrogenSediment Methanea partialtreatment production pressureb

(Pa)

No additions + 1.11 ± 0.16Plus FeCl2C + 1.09 ± 0.14Plus FeSO4C - 0.17 ± 0.16

q +, Indicates detectable methane production; -,indicates methane production was not detectable.

b Mean ± standard error of five observations.c Incubated at least five days, but less than 5 weeks,

with added FeCl2 or FeSO4.

Addition of 5 mM (final concentration) sodiummolybdate to inhibit sulfate reduction in thesulfate-amended sediments resulted in the re-sumption of methanogenesis at a rate compara-ble to that in control sediments (Fig. 1). Thiscorresponded with an increase in the hydrogenpartial pressure which, after an initial accumula-tion, stabilized at partial pressures similar tothose in control sediments. Molybdate had noeffect on the hydrogen partial presure in controlsediments (data not shown).

Since MB maintained their potential to metab-olize hydrogen in sulfate-amended sediments, asuitable inhibitor that would prevent MB fromtaking up added hydrogen but would not inhibithydrogen uptake by SRB had to be found beforekinetic analysis of hydrogen uptake by SRBcould be made. Chloroform (0.003% [vol/vol])inhibited methane production but had no signifi-cant effect on the potential of SRB to metabolizehydrogen, as measured by the rate of sulfatereduction or the rate of hydrogen uptake (Table3).

Sulfate-amended sediments had a higher po-tential for hydrogen uptake than control sedi-ments (Fig. 2, Table 4). The addition of chloro-form to the control sediments resulted in theaccumulation of hydrogen as previously shown(12), but in sulfate-amended sediments a signifi-cant potential for hydrogen uptake remained(Fig. 2, Table 4). The VM of the population thatwas inhibited by chloroform in the sulfate-amended sediments can be calculated as the

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1376 LOVLEY, DWYER, AND KLUG

0

a-LU

LI1uiac

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IE-)

0)

E

TIME (hr)FIG. 1. Methane production rates and hydrogen partial pressures over time in sulfate-amended and control

sediments collected from the A site and incubated at 20°C on a bottle roller. Arrow designates addition ofmolybdate to sulfate-amended sediments. Values are means of duplicate bottles of each treatment and arerepresentative of the results obtained in several similar experiments. Symbols: *-@ and A-A, methaneproduction rates in sulfate-amended and control sediments; 0---0 and A---A, hydrogen partial pressure insulfate-amended and control sediments.

difference between the VM in the sulfate-amend-ed sediments with and without added chloro-form. The value obtained, 0.8 mmol of H2 perliter of sediment per h, was equivalent to the VMof the control sediments. This indicates that thehydrogen uptake potential of the MB populationwas not changed in the sulfate-amended sedi-ment, but that there had been an increase in ahydrogen-consuming potential that was not in-hibited by chloroform.

Half-saturation constants, K, for hydrogenuptake were lower in sulfate-amended sedimentsthan in control sediments (Table 4). When theMB in sulfate-amended sediments were inhibit-ed with chloroform, the resultant K was three-

TABLE 3. Effect of chloroform on the hydrogenuptake potential of methanogens and sulfate reducers

% Inhibition by chloroformaSRB parameter Methae Sulfate

measured behn uftproductionb reduction

Sulfate reduction >94 0.7 (8.9)bHydrogen >% 6.1 (9.8)Cuptakea Mean with standard error in parentheses; n = 3 for

each treatment.b Percent inhibition equals (1 - [rate in sediments

treated with chloroform x rate in control sedi-ments-']) x 100. A minimum estimate for methaneinhibition is shown since there could have been meth-ane production at rates lower than what could bedetected during the incubation period.

c Percent inhibition equals (1 - [sum of the rate ofhydrogen uptake in sediments treated with chloroformand sediments treated with molybdate x uptake rate incontrols-']) x 100.

fold lower than the K in control sediments.When the results of kinetic analyses on sedi-ments collected throughout the summer of 1981from both the A and B site were compiled, theoverall mean K value and 95% confidence inter-val for hydrogen uptake not inhibited by chloro-form was 141 ± 33 Pa (n = 8). This comparedwith the K for MB in control sediments of 597 +186 Pa hydrogen (n = 8).The theoretical progress curves of hydrogen

uptake in sulfate-amended sediments that werecalculated from the two-term Michaelis-Mentenexpression (equation 3) closely correspondedwith those observed experimentally (Fig. 2). Forthese calculations VMSRB and KSRB were takenas the mean values from the chloroform-treated,sulfate-amended sediment. It was assumed thatVMMB was equal to 0.8 mmol of H2 per liter perhour, as calculated above, and that KMB wasequal to the K in control sediments.

DISCUSSIONThe fact that the inhibition of sulfate reduction

in sulfate-amended sediments resulted in an in-crease in the hydrogen partial pressure andmethane production rates to levels found inmethanogenic sediments demonstrated thatwhen sulfate concentrations were not limiting,SRB inhibited methane production by loweringthe hydrogen partial pressure below a thresholdlevel necessary for hydrogen utilization by MB.The inhibition of methane production was notdue to the toxic presence of sulfate or sulfide, aspreviously demonstrated (1, 2, 6, 14, 29) nor tothe depletion of some factor other than theelectron donors necessary for methanogenesis.

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COMPETITION FOR HYDROGEN IN SEDIMENTS 1377

0-ctia.%-

wa:cc(I)wc:a-CNI

1.0 I I I I I Ix

0.8ox

x0.6 v *0

0.4 - xV *xx

0.2 - t 0 *Ot m_0 * x

I I 6T 8 1 2x0- 2 4 6 Ir8 10 12

TIME (hr)FIG. 2. Typical hydrogen uptake progress curves:

Symbols: 0, sulfate, amended sediments; X, sulfate-amended sediments treated with chloroform; 0, con-trol sediments. T represents expected hydrogen partialpressure in sulfate-amended sediments calculatedfrom equation 3 and the appropriate kinetic parame-ters as described in the text.

This conclusion was further supported by thecomparable VM for hydrogen uptake by MB incontrol and sulfate-amended sediments. Thus,the inhibition of methane production by addedsulfate differs from the inhibition by oxygen (31)or nitrogen oxides (3) where the added electronacceptor or a product of its metabolism directlyinhibits MB.The inhibition of methane production at low

hydrogen partial pressures was probably due tothe decreased energy yield from methane pro-duction. The available free energy for methaneproduction from hydrogen was calculated fromthe standard free energy of -139.23 kJ (30) andthe methane, hydrogen, and carbon dioxide par-tial pressures to be -16.3 to -16.8 kJ/mol ofmethane produced in the control sedimentsshown in Fig. 1. The calculated free energy wasapproximately -6.7 kJ/mol of methane pro-

TABLE 4. Kinetic parameters for hydrogen uptakein sediments collected from the A site

Kinetic parametersaSediment type vMb K (Pa)

Control 0.8 ± 0.1 588 ± 70sediments

Sulfate-amended 1.2 ± 0.2 455 ± 111Sulfate-amended 0.4 ± 0.04 175 ± 45

treated withchloroforma Mean and standard error of values from triplicate

progress curves for each treatment. Progress curveswere run concurrently with those shown in Fig. 2.

b Millimoles of hydrogen per liter of sediment perhour.

duced in the sulfate-amended sediments duringthe initial days of the inhibition of methaneproduction and +4.7 kJ at 6 to 7 days after thesulfate addition. Though care must be taken inextrapolating from bulk-phase pool sizes tothose actually experienced by the bacteria, it isclear that the hydrogen partial pressure in thesulfate-amended sediment was sufficiently low-ered to significantly reduce the energy availablefor methane production from hydrogen.The lower hydrogen pool in the sulfate-

amended sediments was associated with thelower overall K for hydrogen uptake and, specif-ically, with the low K for hydrogen uptake bythe bacterial population that was not inhibitedby chloroform. The K for hydrogen uptake inchloroform-treated, sulfate-amended sedimentsis considered to represent the K for the SRBpopulation because: (i) there was no detectablehydrogen uptake in the presence of chloroformin sediments not amended with sulfate; (ii) chlo-roform did not affect hydrogen uptake by SRB;and (iii) molybdate inhibited the hydrogen up-take in sulfate-amended sediments that chloro-form did not inhibit. The K for the MB reportedhere is within the range estimated independentlyfor methanogenic sediments and other methano-genic environments, such as sludge digestorsand the rumen (J. A. Robinson and J. M. Tiedje,submitted for publication). Though there was apossibility of hydrogen uptake by bacteria fer-menting hydrogen and carbon dioxide to ace-tate, the importance of these bacteria in methan-ogenic environments is low relative to methano-gens (5, 13). The conclusion that MB and SRBwere the only two important hydrogen-consum-ing populations is further supported by the ob-servation that the total hydrogen uptake in thesulfate-amended sediments could be predictedby using the K for the sulfate-depleted controlsediment as the k for the population inhibited bychloroform.Under steady-state conditions in environ-

ments, such as sediments, where there is negligi-ble physical removal or dilution of the microbialpopulation, the substrate pool size can be de-scribed by:

S (M XlKS=(VM x ylk) - 1 (4)

where y and k are yield and mortality constantsand K and VM are expressed on a per cell basis(4, 15). Thus, the hydrogen partial pressureshould be dependent solely upon the physiologi-cal characteristics of the hydrogen-consumingpopulations. In the sulfate-amended sediments,the lower SRB K for hydrogen uptake (andpossibly a higher yield and VM per cell) resultedin a lower hydrogen pool. Some of the inhibition

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1378 LOVLEY, DWYER, AND KLUG

of methane production in sulfate-amended sedi-ments may be attributed to the metabolism ofsubstrates by SRB rather than proton-reducingbacteria and the subsequent lower rates of hy-drogen production (6). However, the mainte-nance of a lower hydrogen partial pressure bySRB that consumed hydrogen was the ultimatecause of the complete inhibition of methaneproduction since the hydrogen partial pressurewas independent of the rate of hydrogen produc-tion.The maximum potential rate of substrate up-

take is equally important as the affinity forsubstrate in determining the outcome of compe-tition (9). The slow inhibition of methane pro-duction in Wintergreen Lake sediments amend-ed with 20 mM sulfate can be explained by thesmall initial potential for hydrogen uptake ofSRB. In freshly collected sediments incubatedwith saturating hydrogen, the turnover time for 1mM sulfate (a saturating sulfate concentration)is 204 h (25). Assuming that all of the sulfatereduction was due to hydrogen uptake, thisyields a maximal VM estimate for the SRBpopulation of 19.6 ,umol of hydrogen per liter ofsediment per h. With the estimate that hydrogenis the precursor for approximately 40% of themethane production in these sediments (12), therate of hydrogen production can be calculatedfrom the methane production rate (Table 1) as 64,umol per liter of sediment per h, or threefoldhigher than the SRB VM for uptake. Using theVM and K for the MB, the K for the SRB, andthe hydrogen partial pressure determined in thepresent study, it can be calculated from equation3 that, at saturating sulfate concentrations, SRBwould initially be able to use at most only 10% ofthe total hydrogen consumed by the two popula-tions. Since the in situ sulfate concentration inthese sediments is typically at or below the SRBK for sulfate reduction (24), the limitation ofSRB by sulfate can be expected to lower theSRB maximum potential for hydrogen uptake(22) and result in an in situ hydrogen uptake bySRB that is much less than 10% of the totalhydrogen turnover. This result calculated fromkinetic parameters agrees well with previousconclusions derived from experimental results(12).MB are able to compete successfully with

SRB in Wintergreen Lake sediments despite thelower SRB K for hydrogen uptake because themaximal potential for hydrogen uptake by SRBis limited by sulfate availability. The competi-tion between SRB and MB for acetate is expect-ed to have similar mechanisms as those forhydrogen competition. MB and SRB shouldcoexist in other anaerobic sulfate-containing en-vironments in which the rate of sulfate supplysupports a potential for hydrogen and acetate

uptake by SRB that is lower than the rate ofhydrogen and acetate production.

ACKNOWLEDGMENTSWe thank J. A. Robinson and J. M. Tiedje for helpful

discussions and encouragement during this study. We thankG. M. King for providing the sulfate reduction rates.

This research was supported by National Science Founda-tion grants DEB 78-05321 and DEB 81-09994.

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