Vol. 49, No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1985, p. 944-9480099-2240/85/040944-05$02.OO/OCopyright C) 1985, American Society for Microbiology

Interaction of Acetogens and Methanogens in AnaerobicFreshwater Sediments

J. GWYNFRYN JONES* AND BERNARD M. SIMON

Freshwater Biological Association, The Ferry House, Ambleside, Cumbria LA22 OLP, United Kingdom

Received 9 October 1984/Accepted 23 Jahuary 1985

Anaerobic decomposition processes in the profundal sediments of Blelham Tarn (English Lake District) areoften limited during late summer by the input of organic carbon. The concentration of acetate in the interstitialwater fell from about 100 ,uM (immediately after sedimentation of the spring diatom bloom) to a relativelyconstant value of about 20 ,uM in late summer, during which acetate utilization appeared to be balanced byproduction. Addition of chloroform and molybdate caused an accumulation of cold acetate in large sedimentcores and of [14C]acetate in small cores to which [14C]bicarbonate had been added. In both cases chloroformcaused the greater accumulation, implying that acetoclastic methanogens were the more active consumers. Theconversion of '4Co2 to [14C]acetate was inversely related, with depth, to its conversion to 14CH4. Methanog-enesis from CO2 decreased during late summer, whereas acetogenesis and acetoclastic methanogenesisincreased over the same time period. The production of acetate from CO2 was generally equivalent to less than10% of the acetate carbon utilized but could be as high as 25% of that value. Hydrogen consumption byacetogens could be as high as 50% of that utilized in methanogenesis. The role of acetogenic bacteria inanaerobic processes may therefore be of greater significance in lakes such as Blelham Tarn than in moreeutrophic systems.

The interaction of anaerobic bacteria in the terminalmetabolism of organic carbon in sulfate-lirnited freshwatersediments is now fairly well understood; the reactions maybe summarized as shown in Fig. 1. Organic carbon may bemetabolized to acetate and CO2 via obligate proton reducers,fermentative bacteria, and certain sulfate reducers, but theutilization of the carbon will be complete only in the pres-ence of other acetate utilizers (e.g., sulfate reducers ormethanogens). The reaction to acetate (Fig. 1, reaction 1) isexergonic only in the presence of H2-utilizing sulfate reduc-ers or methanogens, and this process of interspecies hydro-gen transfer has been demonstrated many times in labora-tory cultures since the initial experiments of Bryant et al. (4).The interactions of sulfate reducers and methanogens insulfate-limited sediments have also been investigated widely(11, 23). Sulfate reducers are able to outcompete methano-gens for H2 and acetate in sulfate-poor freshwater sediments(17), presumably reflecting the lower Ks values for thesesubstrates observed in recent experiments on a number oflaboratory strains (14, 20, 21).The metabolic interactions of sulfate reducers mediating

reactions 1 and 2 and methanogens involved in reactions 3and 4 (Fig. 1) are, to a degree, resolved. Little is known,however, about the involvement of acetogenic bacteria(reaction 5), such as Acetobacterium woodii (1) and Clos-tridium aceticum (2), in the terminal metabolism of carbon insediments. Braun et al. (3) reported viable counts of aceto-gens to be ca. 1% of those of methanogens in sludge and lakesediments, and it has been estimated (18) that acetogensaccount for ca. 5% of H2 consumption in the sediments ofhypereutrophic Wintergreen Lake. This lake, and severalother eutrophic water bodies in the United States, undergo acycle in which there is sufficient input of particulate materialto the sediment during the summer to ensure an adequatesupply of organic carbon for the terminal processes ofdecomposition described above. Blelham Tarn profundal

* Corresponding author.

sediments, on the other hand, may not receive sufficientavailable carbon to drive these processes for the duration ofthermal stratification, since sedimentation rates decrease (6)with a consequent sharp decline in methane productionduring late summer (8). During this period, interactionsbetween anaerobic bacteria fluctuate rapidly between phasesof dominance by H2-C02-utilizing methanogens (11) andphases during which acetate-utilizing Desulfotomaculumspp. appear to be the most active component of the popula-tion (9). Under such circumstances, acetogens may assume agreater importance in the acetate budget. Although theactivity of these bacteria has been determined in sedimentslurries from hypereutrophic systems (18) which were incu-bated under artificial atmospheres, we know of no attemptsto more closely reproduce conditions which prevail in thefield (which is essential for the realistic assessment ofreactions dependent on H2 partial pressure). In this paper,we report the conversion of 14CO2 to ['4C]acetate in smallcores of profundal sediment and investigate the possibleinteractions between acetogens and bacteria which consumeacetate.

MATERIALS AND METHODSSampling and sample preparation. Sediment samples were

collected with a Jenkin corer from the profundal zone of theBlelham Tarn (English Lake District: 54o24' N, 2o59' W)during late summner anoxia of the hypolimnion. The tarn is amoderately eutrophic water body; details of the samplingsites and the field methods have been described elsewhere(11). Sediment cores with overlying anoxic water wereopened after the tops of the cores had been placed in anitrogen-filled box. The cores were extruded upward (10)into modified, 30-ml polypropylene syringes. The syringeswere modified by removal of the base of the barrel andchamfering the edge on a lathe. The syringe was sealed at thetop and the piston was replaced immediately, leaving anoxicwater above the relatively undisturbed core. The proceduredid not exclude oxygen on all occasions, but the presence of

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INTERACTIONS OF ACETOGENS AND METHANOGENS

Corganic

I[1]

CH3COOH

[2]1 [5]

[4O,CH4

[3]

C02FIG. 1. Reactions involved in the terminal metabolism of organic

carbon in anaerobic freshwater sediments. The bacteria involvedincluded proton reducers, fermenters and sulfate reducers (reactions1 and 2), methanogens (reactions 3 and 4), and autotrophic aceto-gens (reaction 5).

oxygen was immediately detected by the deposition ofFe(III), the sediments of Blelham Tarn being rich in iron, andby the fact that added acetate was oxidized immediately toCO2. Methanogenesis and acetogenesis were also inhibited.Results from such cores were discarded. Dilutions of sedi-ments were prepared in serum bottles (7) in an attempt toobtain estimates of the most probable number of autotrophic,acetogenic bacteria. The medium was based on one usedpreviously for methanogens (11), with the addition of seleniteand tungstate (NaHSeO3, Na2O4Wo4 * 2H20) at 1 mg liter-'to ensure an adequate supply for the synthesis of formatedehydrogenase (15). The final concentrations of certain vi-tamins was also increased (vitamin B12, pantothenic acid,pyridoxamine to 1 mg liter-', and choline chloride to 2 mgliter-') as precursors in the corrinoid pathway (15). The pHwas adjusted to 7.3 by modification of the phosphate bufferand addition of sodium hydrogen carbonate (sterilized sep-arately by autoclaving under 100% CO2). Sodium acetate andnitrilotriacetic acid were omitted, as acetate was the endproduct to be determined. Sodium sulfide was added as areducing agent at a final concentration of 360 mg liter-'.Resazurin (1 mg liter-' was added as a redox indicator. Thebasic medium was gassed in serum bottles with N2-CO2(95:5, vol/vol) or H2-CO2 (80:20, vol/vol) before autoclaving.Sterilized anoxic vitamin solution and reducing agent wereadded aseptically with sterile syringes. Organic substratesand yeast extract were added to the basal medium as sterileanoxic solutions. Organic substrates were added at a finalconcentration of 1 g liter-'. Purified agar (2%) was added tosolidify the medium for slope and plate preparation. Calciumcarbonate was introduced into solid medium at a final con-centration of 0.025 M to give a fine suspension; acid-produc-ing bacterial colonies gave a clear zone of more-solubleCa(HCO3)2 (1). The acid indicator bromocresol green wasalso used in an agar overlay (3), as were bromothymol blueand methyl red, each at a final concentration of 20 mg liter-'.

Larger-scale experiments involved incubation of wholeJenkin cores containing sediment and overlying water insuch a way as to exclude oxygen and to allow water samplesto be removed after gentle mixing to disperse concentrationgradients (12).

Chemical analyses. Volatile fatty acids were determined bygas chromatography and high-pressure liquid chromatogra-

phy (7). The former was used for cultures and the latter todetermine concentrations in sediment interstitial water. Theagreement between the procedures was good, and theirperformance characteristics have been determined. The useof low concentrations of eluent (0.1 mM H2SO4) permittedthe detection of low (1 1LM) concentrations of acetate, butunder these conditions other organic acids were not sepa-rated satisfactorily (10). Approximately 400 ,ul of untreatedinterstitial water was injected via a Microguard column ontoan HPX87H ion exclusion column (Bio-Rad Laboratories,Richmond, Calif.) with a sampling valve (Rheodyne, Inc.,Berkeley, Calif.). The conductivity detector (type710.9400000; Knauer, Bad. Homburg, Federal Republic ofGermany) was used in conjunction with a Data Mastersystem (Gilson Medical Electronics, Inc., Middleton, Wis.)and an Apple lle microcomputer. Peaks of interest werecollected with a Gilson 201 fraction collector, and their 14Ccontent was determined by liquid scintillation spectrometry.The 14CH4 and 14CO2 contents of the sediments were deter-mined with cores which had been frozen in liquid nitrogen.The cores were sawn into segments, and each segment wasplaced in a 30-ml Teflon open-top septum vial (Tuf Tainers;Pierce & Warriner Ltd., Chester, United Kingdom) to whichwas added 0.5 ml of 10 M NaOH to retain the 14CO2. Thesediment was thawed, and a subsample of the head spacecontaining 14CH4 was injected into a scintillation vial whichcontained 20 ml of a toluene-based fluor. The recovery withthis procedure was consistently about 76%, for which resultswere corrected. The total 14CH4 content of the septum vialwas calculated from Henry's law and published values of theconstant K. For determination of 14CO2, 2 ml of 1.5 M H2SO4was then added to the sediment and further subsamples ofhead space, now containing 14CO2, were injected into aserum bottle containing 0.25 ml of phenethylamine and leftovernight. Excess 14CH4 was flushed out with N2, and thephenethylamine incorporated into the toluene-based fluorand the 14C content were determined by liquid scintillationspectrometry (11).Metabolism of 14C tracers. All radiochemicals were ob-

tained from the Radiochemical Centre, Amersham, UnitedKingdom, and the methods used were essentially those ofJ0rgensen (13), except that a vertical-injection techniquewas used. Anoxic 14C-labeled substrate was injected into thesmall cores with a 10-,lI syringe. The syringe was heldvertically with the piston fixed. The barrel was lowered to fillthe syringe, the needle was inserted to the deepest point ofthe small core, and the barrel was then raised to ensure theinjection of a constant volume (2 pLA cm-') throughout thedepth of the core. The quantity of CO2 added as NaH'4CO3(54 mCi mmol-1; 4 ,uCi cm of depth-') was equivalent to afinal concentration of 20 ,uM, i.e., <1% of the naturalsubstrate concentration. The final concentration of[14C]acetate (58 mCi mmol-'; 0.2 ,uCi cm-') was about 1,uM, which may have been a significant increase towards theend of summer, when the natural concentration of thissubstrate fell to <20 ,uM. The rate of conversion of acetateto CO2 and CH4 may have been stimulated as a result, butthis could not be said of the conversion of 14CO2 to acetate.The final concentrations of [U-_4C]glucose (296 mCi mmol-1;0.2 ,Ci cm-') and [U-_4C]glycine (110 mCi mmol-'; 0.1 ,uCicm-1) were 0.19 and 0.25 pFM, respectively. The ratesobtained with these additions have been treated as Vmaxvalues, since the levels of the natural substrates wereanalytically undetectable. The final concentrations of inhib-itors used were 2 mM Na2MoO4 and 120 puM CHCl3, whichwere added to small cores and other systems 2 h before 14C

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946 JONES AND SIMON

tracer injection. The concentration of molybdate was chosenas a result of earlier experiments on its effect on organismsother than sulfate reducers (11, 16) in freshwater sediments.Chloroform was added as a saturated solution in water, theconcentration of which was adequate to inhibit methanogen-esis in both Jenkin cores and small cores (16). Cores wereincubated for 1 h at 10°C before determination of 14C-labeledproducts as described above.The data presented are means of triplicate determinations.

The coefficient of variation at any core depth on a given daywas of the order of 5%. If samples were stored overnightbefore analysis, then this coefficient could increase to 9%.

RESULTSIn water bodies such as Blelham Tarn, where the supply of

particulate material to the sediment may limit anaerobicdecomposition during late summer, a decline in acetateconcentration is observed as summer progresses. The valuesobtained during 1983 (Table 1) show that the concentrationin the sediment interstitial water was highest in June (ca. 100,uM, after sedimentation of the Spring bloom ofphytoplankton) and then decreased to relatively stable val-ues during late summer. Acetate concentrations generallydecreased with depth of sediment. Addition of '4C-labeledglucose, glycine, and CO2 to small cores during late summerresulted in estimated turnover times to acetate of 147, 73,and 830 h, respectively. Glucose and glycine were, however,analytically undetectable in the interstitial water at this timeof year, and therefore the trace addition must have affectedthe available concentration, possibly resulting in a metabolicrate which approximated maximum velocity. Despite this,when the pool size of inorganic carbon was taken intoaccount, it became clear that the conversion of C02, pre-sumably by autotrophic acetogens, could provide a signifi-cant quantity of acetate (Table 2). Estimates of the mostprobable number of acetogenic bacteria in these sedimentswere not reproducible, and the sediments failed to yield purecultures of acetogens which grew on a mineral medium andutilized H2 and CO2. Acetogenesis could be detected at asediment dilution of 10-4 on the mineral medium, but largerquantities of acetate were obtained when the medium wassupplemented with glucose, glycine, or yeast extract. Thequantity of acetate produced was an approximate linearfunction of yeast extract concentration over the range 0.01to 1.0 g liter-1.Methanogens were considered to be quantitatively more

important than sulfate reducers as consumers of acetate,since the addition of chloroform (an inhibitor of methano-gens) resulted in a greater and more rapid accumulation ofacetate in sediment cores (Fig. 2) than did the addition of

TABLE 1. Acetate concentration in the interstitial water ofBlelham Tarn profundal sediments during late summer 1983

Sediment Concn (,uM) on:'depth (cm) 2 June 29 June 4 August 6 September 4 October

0-1 58 96 29 23 191-2 36 78 22 25 92-3 28 80 22 21 113-4 33 34 20 23 84-5 24 35 20 14 105-6 42 32 17 17 8

"The coefficient of variation within cores was <5%; that between coreswas <12%.

TABLE 2. Rates of acetate production in the top 4 cm ofBlelham Tarn profundal sediment during late summer

Date of Acetate produced (nmol g-' [dry wt] day-') from':substrateaddition Glucose Glycine CO,

27 July 1.15 (0.7) 0.95 (0.86) 41.8 (19.4)28 August 2.13 (1.24) 1.58 (0.96) 145.5 (113.4)

a Values are means and, in parentheses, 95% confidence limits; n = 4.

molybdate (an inhibitor of sulfate-reducing bacteria). Theseresults were confirmed by the observation of a more rapidturnover of [2-14C] acetate to 14CH4 (ca. 2.5 h) than of [1-14C]acetate to "'CO2 (ca. 3.2 h) in small-core experiments.

Although acetate is derived from many sources in fresh-water sediments, the purpose of this study was to determinewhether autotrophic acetogens were of any significance andwhether they were involved in metabolic interactions withmethanogens and sulfate reducers. This was achieved byintroducing trace quantities of 14C-labeled CO2 and acetateinto small sediment cores and determining the net rates ofacetate formation and utilization in the presence and ab-sence of inhibitors specific for methanogens and sulfatereducers. At the end of July, the rate of conversion of CO2 toCH4 was high, whereas the rate of conversion to acetate waslow (Fig. 3a); by the end of August, methanogenesis fromCO2 was reduced significantly (P < 0.05) in the 0- to 1-cmand 1- to 2-cm samples, and acetogenesis increased (Fig.3b). This increase was significant (P < 0.05) only in thesurface sediment. At the same time, acetoclastic methano-genesis also increased. The depth distribution pattern ofacetogenesis at the end of August was the inverse of that formethanogenesis by CO2 reduction. In the presence of chlo-roform, the quantity of [14C]acetate that accumulated wasgreatly increased, and a similar effect, although slightly lessmarked, was observed with molybdate (Fig. 3c). Once again,the production of acetate was greatest at the sedimentsurface. When the rates of acetate production (from C02)and its consumption by oxidation and methanogenesis werecalculated, it became evident that, although C02-utilizingacetogens generally accounted for less than 10% of theacetate carbon utilized, values as high as 25% were observed

2 500 _

C

0

° 250 / _ S oo0Dp250 0 ~~~~~~~~0

o0 10 20 30

Incubation time (d)FIG. 2. Effect of inhibitors of methanogens and sulfate-reducing

bacteria on the accumulation of acetate in Jenkin cores of BlelhamTarn profundal sediment. The coefficient of variation on quintupli-cate determinations was <8%. Symbols: 0, control; C, 2 mMmolbydate; *, 120 ,uM chloroform.

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INTERACTIONS OF ACETOGENS AND METHANOGENS

0

2

_-- 3

0)

*E3a)

0-

1-

2

3.

0 0.5 1.0 1.5

Conversion rates (u mol g-1 d-1)FIG. 3. Rates of conversion of "CO2 to acetate (0) and to CH4

(0), [2-14C]acetate to CH4 (U), and [1-14C] acetate to CO2 (O) duringJuly and August and of t4CO2 to acetate in the presence ofchloroform (A) and molybdate (A) during August. Experimentswere performed on small sediment cores taken from the profundalzone of Blelham Tarn during late summer anoxia.

on occasion (Table 3). When these values were converted tothe equivalents of hydrogen consumption by acetogens andC02-utilizing methanogens, it became clear that the formercould consume a quantity of H2 equivalent to 50% of thatused by methanogens.

DISCUSSION

The rapid decline in methanogenesis during late summerhas been observed in Blelham Tarn over several seasons (8,11); during this period, fluctuations in the metabolic relation-ships between methanogens and sulfate-reducing bacteriaoccur (9, 11). The fact that decomposition may becomelimited by the input of organic carbon and that acetateconcentrations are much lower distinguishes this site fromhypereutrophic systems such as Wintergreen Lake (19).

Gradients of acetate concentration, such as those reportedfor Lake Vechten (5), where concentrations fell at thesediment surface (implying net utilization or loss at thesediment water interface), were not observed. The concen-tration of acetate in Blelham Tarn was highest at thesediment surface in early summer, suggesting that it wasderived at this time from the decomposition of sedimentedorganic matter. By late summer the concentration had fallen,and from August to October the values observed at thesurface were not significantly different from those at greaterdepths. Under such circumstances, sources of acetate otherthan incoming organic carbon may be of significance, and itwas for this reason that we decided to investigate the role ofC02-reducing acetogens in Blelham Tarn.The concentration of acetate remained relatively stable

during late summer, although the processes involved in itsutilization (methanogenesis and oxidation) continued. Theseprocesses must therefore have been balanced by thoseresponsible for acetate production, which might includefementation (with or without the involvement of interspeciesH2 transfer), deamination of glycine (22), and acetogenesisfrom CO2 and H2. Although glucose and glycine were bothconverted to acetate faster than was C02, the relative poolsizes of these components indicated that the reduction ofCO2 by H2 may be more important than had been appreci-ated. The bacteria which mediate this reaction include A.woodii (1) and C. aceticum (2), but we were unable to isolatethese or any other bacteria for which we were satisfied thatthe major source of acetate was CO2 and H2. The relation-ship between acetate production and the initial concentra-tion of yeast extract in the medium suggested that deamina-tion of amino acids may be responsible for some of theacetate produced. We concluded that the incubation periodemployed (4 to 6 weeks) was too short for the initial isolationof autotrophic acetogens on the medium used.

Inhibitor studies with molybdate and chloroform in bothJenkin cores and with radiotracers in small cores indicatedthat methanogens consume more acetate during the latesummer than do sulfate-reducing bacteria. The quantity ofacetate which accumulated in such experiments must repre-sent that which was biologically available. The adsorptionand binding of acetate is not as great in Blelham Tarnsediments as that observed in marine systems in particular,and its availability as a substrate to bacteria is consideredelsewhere (10).The depth distribution of CO2 conversion to acetate was

the inverse of that for its conversion to CH4, suggesting a

TABLE 3. Comparison of carbon and hydrogen flow during the formation and utilization of acetate and the reduction of CO, in theprofundal sediments of Blelham Tarn

Flow (nmol g [dry wtJ-' day-') of:Date and Carbon Hydrogen

sediment depth(cm) CO, CH3COOH CH3000H CO2 A/B (%) CO2 CH3COOH CO2 CH4 A/B (%)

(A) and CH4 (B) (A) (B)

27 July 19820-1 31 730 4.3 124 7,150 1.71-2 23 512 4.5 92 5,160 1.82-3 49 720 6.8 196 2,800 7.03-4 66 944 7.0 264 1,520 17.4

28 August 19820-1 311 1,214 25.0 1,244 2,400 51.81-2 76 1,057 7.2 304 3,560 8.22-3 68 1,014 6.7 272 3,080 8.83-4 127 1,116 11.4 508 2,240 22.7

a. 27 July 1982

DE 0 /D\ \OI \ \6o m cb \

b. 28 August 198200 "

o/ IC 0

b DC. 28 August 1982

A

_ IA A

~II

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948 JONES AND SIMON

the inverse of that for its conversion to CH4, suggesting apotential for competition between C02-utilizing acetogensand methanogens. Similarly, an increase in acetogenesiswith time accompanied a decline in methane productionfrom CO2 but an increase in acetoclastic methanogenesis.Addition of molybdate to these samples also stimulatedmethanogenesis, suggesting that the sulfate-reducing popu-lation at this time of year may be dominated by acetateutilizers, as reported previously (9). These results providefurther evidence that the metabolic interactions betweenanaerobes in Blelham Tarn sediment are not only complex,but also fluctuate quite rapidly during late summer.The absolute value for hydrogen consumption by aceto-

gens (3.6 p.mol liter-' h-') was not much greater than thatfound in Wintergreen Lake (18) (2.7 ,umol liter-' h-1), but interms of the overall H2-consuming processes, the acetogensin Blelham Tarn could consume an amount of H2 equivalentto 50% of that used by methanogens (assuming that 4 mol ofH2 is required to reduce 1 mol of CO2 [22]). Hydrogenconsumption by acetogens was therefore of greater signifi-cance in the terminal metabolism of carbon in Blelham Tarn.This assertion should not be made, however, without someexplanation of the assumptions and calculations used in thisstudy. Rates were calculated from the product of the turno-ver constant and the natural concentration of substrate. ForC02, this concentration ranged between 2.5 and 5.0 mM,whereas the acetate concentration fell significantly duringlate summer to less than 20 p.M, and on occasions thequantity of 14C-labeled acetate added may have significantlyaltered the sediment pool size (10). This may have resultedin an overestimate of the rate of acetate consumption. Therates of acetate production from CO2 assumed that 2 mol ofCO2 is incorporated into each mole of acetate; however,pathways other than direct reduction of CO2 exist in whichonly one acetate carbon is derived from CO2 (15). In additionto these factors, chloroform was used in certain experimentsto inhibit methanogenic bacteria. Chloroform is also re-ported to inhibit the corrinoid pathway of some acetogens(15), and therefore the acetate which accumulated in theseexperiments may have been produced from other auto-trophic routes, or the inhibition of the corrinoid pathwaymay have been incomplete at the concentration of chloro-form used. With these comments in mind, and in light of thefact that experiments were conducted under conditionswhich attempted to maintain natural partial pressures of H2(i.e., artificially high H2 atmospheres were not used), therelative importance of CO2 incorporation into acetate couldnot have been overestimated.The results indicate that, in lakes such as Blelham Tarn,

where organic carbon input to profundal sediments may belimiting in late summer, acetogenic bacteria may play a moresignificant role in interactions with other anaerobes than thatobserved in more eutrophic systems.

ACKNOWLEDGMENTSWe thank Catherine A. Simpson and Morag Ferrie for technical

assistance and Joyce Hawksford, who typed the manuscript.This research was financed by the Natural Environment Research

Council.

LITERATURE CITED1. Balch, W. E., S. Schoberth, R. S. Tanner, and R. S. Wolfe. 1977.

Acetobacterium, a new genus of hydrogen-oxidizing, carbondioxide-reducing, anaerobic bacteria. Int. J. Syst. Bacteriol.27:355-361.

2. Braun, M., F. Mayer, and G. Gottschalk. 1981. Clostridiumaceticum (Wieringa), a microorganism producing acetic acidfrom molecular hydrogen and carbon dioxide. Arch. Microbiol.128:288-293.

3. Braun, M., S. Schoberth, and G. Gottschalk. 1979. Enumerationof bacteria forming acetate from H2 and CO2 in anaerobichabitats. Arch. Microbiol. 120:201-204.

4. Bryant, M. P., L. L. Campbell, C. A. Reddy, and M. R. Crabill.1977. Growth of Desulfovibrio in lactate or ethanol media low insulfate in association with H2-utilizing methanogenic bacteria.AppI. Environ. Microbiol. 33:1162-1169.

5. Cappenberg, T. E., K. A. HordiJk, G. J. Jonkheer, and P. M.Lawen. 1982. Carbon flow across the sediment-water interfacein Lake Vechten, The Netherlands. Hydrobiologia 91:161-168.

6. Jones, J. G. 1976. The microbiology and decomposition ofseston in open water and experimental enclosures in a produc-tive lake. J. Ecol. 64:241-279.

7. Jones, J. G., S. Gardener, and B. M. Simon. 1984. Reduction offerric iron by heterotrophic bacteria in lake sediments. J. Gen.Microbiol. 130:45-51.

8. Jones, J. G., and B. M. Simon. 1980. Decomposition processesin the profundal region of Blelham Tarn and the Lund tubes. J.Ecol. 68:493-512.

9. Jones, J. G., and B. M. Simon. 1984. The presence and activityof Desulfomaculum spp. in sulphate limited freshwater sedi-ments. FEMS Microbiol. Lett. 21:47-50.

10. Jones, J. G., and B. M. Simon. 1984. Measurement of microbialturnover of carbon in anoxic freshwater sediments: cautionarycomments. J. Microbiol. Methods 3:47-55.

11. Jones, J. G., B. M. Simon, and S. Gardener. 1982. Factorsaffecting methanogenesis and associated processes in the sedi-ments of a stratified eutrophic lake. J. Gen. Microbiol. 128:1-11.

12. Jones, J. G., B. M. Simon, and R. W. Horsley. 1982. Microbio-logical sources of ammonia in freshwater lake sediments. J.Gen. Microbiol. 128:2823-2831.

13. J0rgensen, B. B. 1978. A comparison of methods for thequantification of bacterial sulfate reduction in coastal marinesediments. I. Measurement with radiotracer techniques. Geomi-crobiol. J. 1:11-27.

14. Kristjansson, J. K., and P. Schonheit. 1983. Why do sulfate-re-ducing bacteria outcompete methanogenic bacteria for sub-strates? Oecologia 60:264-266.

15. Ljungdahl, L. G., and H. G. Wood. 1982. Acetate biosynthesis,p. 165-202. In D. Dolphin (ed.), B12. Wiley Interscience, NewYork.

16. Lovley, D. R., and M. J. Klug. 1982. Intermediary metabolismof organic matter in the sediments of a eutrophic lake. Appl.Environ. Microbiol. 43:552-560.

17. Lovley, D. R., and M. K. Klug. 1983. Sulfate reducers canoutcompete methanogens at freshwater sulfate concentrations.Appl. Environ. Microbiol. 45:187-192.

18. Lovley, D. R., and M. J. Klug. 1983. Methanogenesis frommethanol and methylamines and acetogenesis from hydrogenand carbon dioxide in the sediments of a eutrophic lake. Appl.Environ. Microbiol. 45:1310-1315.

19. Molongoski, J. J., and M. J. Klug. 1980. Anaerobic metabolismof particulate organic matter in the sediments of a hypereu-trophic lake. Freshwater Biol. 10:507-518.

20. Robinson, J. A., and J. M. Tiedje. 1984. Competition betweensulfate reducing and methanogenic bacteria for H2 under restingand growing conditions. Arch. Microbiol. 137:26-32.

21. Schonheit, P., J. K. Kristjansson, and R. K. Thauer. 1982.Kinetic mechanism for the ability of sulphate reducers toout-compete methanogens for acetate. Arch. Microbiol. 132:285-288.

22. Thauer, R. K., K. Jungermann, and K. Decker. 1977. Energyconservation in chemotrophic anaerobic bacteria. Bacteriol.Rev. 41:100-180.

23. Winfrey, M. R., and J. G. Zeikus. 1977. Effect of sulphate oncarbon and electron flow during microbial methanogenesis infreshwater sediments. Appl. Environ. Microbiol. 33:275-281.

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