Vol. 45, No. 6APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1983, p. 1848-18530099-2240/83/061848-06$02.00/0Copyright © 1983, American Society for Microbiology

Metabolism of Acetate, Methanol, and Methylated Amines inIntertidal Sediments of Lowes Cove, Mainet

GARY M. KING,'* M. J. KLUG,2 AND D. R. LOVLEY3

I. C. Darling Center, University of Maine, Walpole, Maine 045731; W. K. Kellogg Biological Station,Michigan State University, Hickory Corners, Michigan 490602; and Department ofAnaerobic Microbiology,

Virginia Polytechnic Institute and State University, Blacksburg, Virginia 240613

Received 20 December 1982/Accepted 31 March 1983

The fates and the rates of metabolism of acetate, trimethylamine, methylamine,and methanol were examined to determine the significance of these compounds asin situ methane precursors in surface sediments of an intertidal zone in Maine.Concentrations of these potential methane precursors were generally <3 ,uM,with the exception of sediments containing fragments of the seaweed Ascophyl-lum nodosum, in which acetate was 96 ,uM. [2-14C]acetate turnover in all sampleswas rapid (turnover time <2 h), with 14Co2 as the primary product. [14C]trimeth-ylamine and methylamine turnover times were slower (>8 h) and were character-ized by formation of both 14CH4 and 14C02. Ratios of 14CH4/14CO2 from[14C]trimethylamine and methylamine in uninhibited sediments indicated that a

significant fraction of these substrates were catabolized via a non-methanogenicprocess. Data from inhibition experiments involving sodium molybdate and 2-bromoethanesulfonic acid supported this interpretation. [14C]methanol was oxi-dized relatively slowly compared with the other substrates and was catabolizedmainly to 14CO2. Results from experiments with molybdate and 2-bromoethane-sulfonic acid suggested that methanol was oxidized primarily through sulfatereduction. In Lowes Cove sediments, trimethylamine accounted for 35.1 to 61.1%of total methane production.

In marine sediments, competition for sub-strate has been considered a major factor limit-ing methanogenesis (1-3, 7-9, 14, 15, 18, 22, 25).Sulfate-reducing bacteria (SRB) out-competemethane-producing bacteria (MPB) for both H2and acetate over a wide range of sblfate concen-trations because thermodynamic and kineticconsiderations favor sulfate reduction (10, 10a,21). Studies of competition for acetate haveindicated that acetate hydrolysis to CO2 andCH4 occurs only in sulfate-depleted sedimentsand, by inference, that the reduction of CO2 byH2 is a significant source of methane in marinesediments (2, 9, 14, 18, 20, 25). Even thoughSRB have a competitive advantage, H2 uptakeby MPB is apparently not entirely excluded.Most studies of marine sediments have not

considered methane production from substratesother than H2 or acetate. However, Senior et al.(22) have suggested that some unknown sub-

t Contribution no. 163 of the Darling Center, 83-01 of theMaine Benthic Research Group, 500 of the Kellogg BiologicalStation, and 10,730 of the Michigan Agricultural ExperimentStation.

strate(s) may be more important than H2 inmarsh sediments. Oremland et al. (16) andOremland and Polcin (17) have proposed thattrimethylamine (TMA) and methanol are non-competitive methane precursors in salt marshsoils and San Francisco Bay sediments. Obser-vations by Oremland et al. (16) indicate thatTMA and methanol metabolism could accountfor measured rates of methane production. Win-frey and Ward (26) have also noted that methyl-amine (MA) is readily converted to methane andthat the metabolism of this compound may be ofgreater importance than H2 for methanogenesis.The metabolism of methylated amines in marinesediments could be especially significant be-cause high concentrations of these compoundsare produced by marine organisms for osmoreg-ulation (28).We report here a study of acetate, methanol,

MA, and TMA metabolism at in situ concentra-tions in surface sediments of an intertidal mudflat in Maine. Results indicate that acetate andmethanol were catabolized primarily by SRB,that TMA and MA may have been degraded byboth SRB and MPB, and that TMA was asignificant source of methane (35 to 61%).

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METHANE PRECURSORS IN MARINE SEDIMENTS 1849

MATERIALS AND METHODS

Sediments were obtained from the intertidal zone ofLowes Cove, Maine (43°56'N, 69°35'W). Tides in thisregion are semidiurnal, with an amplitude of 2 to 3 mand a salinity of 30 ppt (30 '/O.). The intertidal sedi-ments of Lowes Cove consist of fine silts and clayswith an organic content of 1 to 2%. Aspects of thesediment chemistry and biology have been describedelsewhere (P. T. Rahaim, M.S. thesis, University ofMaine, Orono, 1980).

Surface sediments (0 to 10 cm depth) of two typeswere collected from the midcove region. In one type,designated here as bare sediments (BS), the upper 5cm appeared oxidized, and the lower 5 cm was morereduced, as indicated by the presence of iron sulfides.BS were also characterized by low dissolved sulfide(<10 FM) and relatively low rates of sulfate reduction(<100 nmol/cm3 per day) and methanogenesis (Table1; G. M. King, unpublished data). Little or no sulfatedepletion was observed over the upper 10 cm in BS.The second sediment type, designated as Ascophyllumnodosum sediments (ANS), was obtained from shal-low depressions within the BS. These depressionsusually contained a lens of water (<10 cm depth) atlow slack tide and partially buried fragments of therockweed A. nodosum. ANS were visibly black at thesediment surface and were often colonized by sulfide-oxidizing bacteria which formed a white mat on thesurface. Dissolved sulfide (-1 mM) and rates of sul-fate reduction (>1 MLmol/cm3 per day) "id methano-genesis were relatively high at ANS sites (Table 1;King, unpublished data). Sulfate concentrations inANS were generally less than in BS but were still -15mM at 10 cm. BS were characterized by a diversefauna consisting of a variety of decapods and mol-luscs; this faunal assemblege was notably absent fromANS sites (D. S. Shaw, unpublished data).

Surface sediments from BS and ANS sites werecollected by using 10-cm inside diameter coring tubes.Cores were placed in an anaerobic glove bag, and theupper 10 cm of the sediment was removed and slur-ried (1:1) with 1-,um-ffltered, deoxygenated seawater(30 0/00) This treatment would have resulted in little orno increase in sulfate concentrations in BS and onlymoderate increases in ANS. Because only moderate, ifany, sulfate depletion was observed in these sedi-ments, it was presumed that no mixing of sulfate-reducing and methanogenic layers occurred. Aliquots(10 cm3) of the slurry were dispensed into 15-miVacutainer tubes or Hungate pressure tubes (BellcoGlass, Inc.) which were subsequently flushed with 02-free 100% N2 and sealed. Then 0.3 ml of a solutioncontaining one of the following substrates was injectedby needle and syringe into each of 15 replicate tubescontaining BS or ANS: [14C]TMA, -0.4 ,Ci/ml (5.0mCi/mmol; New England Nuclear); [14C]MA, -0.3pCi/mi (51.8 mCi/mmol; New England Nuclear);[14C]methanol, -0.2 pCi/ml (56.9 mCi/mmol; Amer-sham); [2-14C]acetate, -2 pCi/ml (51 mCi/mmol, NewEngland Nuclear). Triplicate tubes of BS and ANScontaining the first three of the above-mentioned solu-tions were incubated at 20°C (field temperature) for 0,1, 3, 6, or 12 h. Triplicate tubes, as described above,containing [2-14C]acetate were incubated at 20°C for 0,0.5, 1, 2, or 4 h. Reactions were terminated byinjecting with a needle and syringe 1 ml of a 20%solution of glutaraldehyde.

Total rates of methane production for the ANS andBS slurries were measured by using quadruplicates ofeach sediment type to which no radioisotopes wereadded. A 0.5-ml volume of the gas phase of each ofthese control samples was analyzed for methane byusing a Varian 1440 flame ionization gas chromato-graph (Varian Instruments, Inc.) fitted with a stainlesssteel column (0.32 cm outer diameter by 1 m) contain-ing Porpak Q (Waters Associates, Inc.). The columnwas operated at 75C with a carrier gas of N2 at 30ml/min. Control sediments were incubated for 12 h at20°C for rate determinations.

In similar experiments, 10 cm3 of a 1:1 slurry ofANS was preincubated for 24 h with sodium molyb-date (20 mM final concentration) or 2-bromoethanesul-fonic acid (BES; 27 mM final concentration) to ensureeffectiveness of the inhibitors. Afterwards, 0.3 ml ofthe above-mentioned solutions of radioisotopes wasinjected by needle and syringe. Three replicates ofeach substrate plus inhibitors were incubated at 20°Cfor 6 h. Activity was terminated as described above.

Production of 14CH4 and "4CO2 was determined bysubsampling the gas phases of the tubes with a needleand syringe. Samples (1.00ml) were analyzed with aVarian 3760 gas chromatograph fitted with a stainlesssteel column (0.32 cm outer diameter by 1 m) contain-ing Porpak N (Waters). The column was operated at40°C with a flow rate of N2 of 20 mllmin. 14CH4 and14CO2 were eluted with retention times of 1.3 and 4min, respectively, and were passed through a flameionization detector to oxidize 14CH4. Exhaust from thedetector was bubbled through a series of two scintilla-tion vials containing 7 ml of 0.5 N KOH to trap 14CO2originally in the sample and "4CO2 originating from any14CH4. A separate set of traps was used for 14CH4 and14CO2; trap sets were selected by using a switchingvalve. A 7-mi volume of Aqueous Counting Scintillant(Amersham Searle) was added to each vial, and radio-activity was determined by using a Beckman LS 7500scintillation counter (Beckman Instruments, Inc.). Re-covery of label in the traps was >800o. The distribu-tion of 14CO2 and H14CO3- between the gas anddissolved phases of the sediment samples was deter-mined by adding known amounts of H14CO3- to tubesas described above and then measuring 14CO2 in theheadspace after an 8-h equilibration period. The ob-served distribution of 1'CO2 and H14CO3- was used tocorrect headspace 14CO2 for total "4CO2.

Acetate uptake in ANS and BS was determined bymeasuring the activity of the [2-14C]acetate remainingin samples to which [2-14C]acetate had been added. A1.0-ml volume of the interstitial water from each of thesediment samples was acidified to remove 14CO2 andthen was mixed with 3 ml of Aqueous CountingScintillant for liquid scintillation counting. Previousstudies involving radio-gas chromatography as de-scribed by Lovley and Klug (11) indicated that addedacetate was converted only to gaseous end productsand not to other soluble metabolites.The recovery of added label in the preceding experi-

ments averaged >95% for the various substrates inboth ANS and BS. Recovery was determined from thesum of 14CO2, 14CH4, and dissolved 14C measured atthe final incubation point. No "4CO2 or 14CH4 wasmeasured for controls containing glutaraldehyde.The potential response of ANS to substrate addi-

tions was determined by incubating 50 cm3 of a 1:1slurry of the 0- to 10-cm depth interval in 300-ml

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1850 KING, KLUG, AND LOVLEY

biological oxygen demand bottles containing an atmo-sphere of 02-free 100% N2. Sediments were incubatedin the dark for 48 h at 20°C. Basal rates of methaneproduction during this period were determined byremoving samples (0.5 ml) of the gas phase of thebottles and analyzing methane with the Varian 1440flame ionization detector previously described. After48 h, solutions of TMA, MA, methanol, or acetatewere added to a final concentration of 1 mM. Rates ofmethane production were determined for triplicates ofeach substrate during an additional 48-h incubationperiod at 20°C.

Concentrations of methanol and methylamines weredetermined by gas chromatography, using a Varian3740 with flame ionization detectors. Interstitial waterfrom both ANS and BS was collected by centrifuga-tion and was shipped frozen to the Kellogg BiologicalStation, Hickory Corners, Mich., for analysis (12).Briefly, methanol was determined after distillation andinjection of the distillate onto a 2-m glass column (2-mm inside diameter) containing Porpak Q. Recoveryof added methanol was 73%. MAs were analyzed byevaporating to dryness aliquots of acidified interstitialwater containing 20 mg of (NH4)2SO4. A volume of 10N NaOH (10 ml) was injected into sealed bottlescontaining the residue from evaporation. MAs in theheadspaces of the bottles were assayed by injection of1 ml of the headspace gas into a 2-m glass column (2-mm inside diameter) containing Carbopak B-4%Carbowax 20M-0.8% KOH (Supelco, Inc.) operatedat 85°C with helium as a carrier. Recoveries of MAswere approximately 70%.

Acetate concentrations in interstitial water weremeasured at the I. C. Darling Center, Walpole, Maine,using a Varian 3760 gas chromatograph. Aliquots ofinterstitial water (1 to 2 ml) were acidified (pH <2)with H3PO4 and distilled under vacuum (23). Distillatewas injected onto a 1-m glass column (4-mm insidediameter) containing Carbopak B-4% Carbowax 20M-10%o H3PO4 (Supelco). Absolute recovery of acetatewas >85%, but concentrations were determined rela-tive to an internal standard, isovaleric acid, the recov-ery of which also exceeded 85%.

RESULTSThe time course of 14CO2 formation from [2-

14C]acetate was hyperbolic (Fig. 1) for bothANS and BS. Added [2-14C]acetate was con-verted primarily to 14Co2. About 92 and 46% ofthe added acetate was recovered as 14CO2 within4 h in ANS and BS, respectively. These relativedifferences in 14CO2 production were evident inrates of [2-14C]acetate uptake (Fig. 1). De-creases in [2-14C]acetate were approximatelymirrored by increases in 14CO2 production, andturnover times calculated from either acetateuptake or end product formation were similar.Acetate turnover times based on 14CO2 produc-tion at 1 h were 1.8 and 2.9 h for ANS and BS,respectively.The time courses of 14CH4 and 14CO2 from

both [14C]TMA and [14C]MA were also hyper-bolic (Fig. 2 [BS data not shown]). The percentcatabolism of these substrates in ANS was lessthan that of acetate but was similar to acetate

100

0

wa<50

U-0

0 2 3 4

TIME (hrs)FIG. 1. Uptake of [2-14C]acetate and production

of 14CO2 in slurries of ANS and BS. Each point isthe mean of triplicate samples. Representative stan-dard error bars are shown for ANS (± one standarderror); error bars smaller than the symbols are notshown. Symbols: *, [2-14C]acetate; *, 14CO2; -ANS;----, BS.

catabolism in BS. Average total turnover timesover the 0- to 6-h incubation period were 8.5 and9.0 h for TMA and MA respectively, in ANS,and 12.6 and 8.7 h, respectively, in BS. Ratios of14CH4/14CO2 at the termination of the experi-ments were relatively low (c2) for [14C]TMAand [14C]MA in both sediment types (Table 1).End product formation from [14C]methanol

differed somewhat from that of other substrates.Both 14CH4 and 14CO2 were produced rapidly,within 1 to 3 h, but no significant changes wereobserved thereafter (Fig. 2). Turnover times to14CH4 were long relative to other substrates

100

50o 50 _ ,__ _- -

0 3 6 9 12TIME (hrs)

FIG. 2. Production of 14CO2 and 14CH4 from[14C]methanol, [14C]TMA, and [14C]MA. Each point isthe mean of triplicate samples of ANS slurries. Repre-sentative standard error bars are shown for productsfrom ['4C]TMA (± one standard error); error barssmaller than the symbols are not shown. Symbols: A,products of [14C]methanol; 0, products of [14C]TMA;*, products of [14C]MA; -, 14CH4; ----, 14CO2.

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METHANE PRECURSORS IN MARINE SEDIMENTS 1851

TABLE 1. Methane production and patterns ofsubstrate concentrations and metabolism in slurries

of Lowes Cove sediments% oftotal

Sub- Methane 14CHX14CO2 meth-Site Substrate strate produc- ane

concna tionb ratio pro-duc-tiond

BS Acetate 11.0 <0.001 <0.003 0.5Methanol 3.3 0.003 0.01 2.4TMA 2.2 0.036 1.95 61.1MA ND 0.028 0.51

ANS Acetate 96.0 <0.001 <0.001 5.5Methanol 1.5 <0.001 0.19 0.1TMA 2.4 0.091 2.04 35.1MA ND 0.057 1.34

a Expressed as micromoles per liter of the sedimentslurries described in the text. ND, Not detected.

b Methane production is expressed as the fraction of14C substrates converted to 14CH4 and 14CO2 viamethanogenesis per hour. Values were determined byusing measured 14CH4 and 14CO2 calculated fromtheoretical stoichiometries for each substrate; valueswere calculated for the 0- to 6-h incubation period.

c Ratio of 14CH4/14CO2 after 6 h of incubation.d Average rates (n = 4) of total methane production

in BS were 0.29 nmol/cm3 per h; average rates (n = 4)in ANS were 1.4 nmollcm3 per h.

(Table 1), though total turnover times were lessthan for [14C]TMA and [14C]MA. Ratios of14CH4/14CO2 from [14C]methanol were quite low(Table 1), and there was no trend for valuesincreasing with time.

Concentrations of the various substrates in-vestigated were all relatively low, with the ex-ception of acetate in ANS (Table 1). MA was notdetectable in BS or ANS. The observed concen-trations and rates of 14CH4 production wereused to calculate the contribution of each sub-strate to total methane production measured forthe ANS and BS slurries. Methane productionfrom acetate and methanol was negligible in bothsediment types (Table 1). Actual contributionsfrom MA could not be calculated because MAwas not detected; potential contributions basedon the lower limit of detection, 1 ,uM, were<10%. The percentage of total methane produc-tion derived from TMA varied from 35.1% inANS to 61.1% in BS.

Addition of sodium molybdate (20 mM finalconcentration) resulted in an almost completeinhibition of acetate oxidation and an increase inthe ratio of 14CH4/14CO2 from [2-14C]acetate;the total fraction of acetate catabolized was-1% (Table 2). 14CH4/14CO2 ratios from[14C]TMA, [14C]MA, and [14C]methanol alsoincreased markedly in the presence of molyb-

date, as did the total fraction of substratescatabolized. The addition of BES (27 mM finalconcentration) resulted in a significant decreasein 14CH4 formation and 14CH4/14CO2 ratios fromall substrates (Table 2). With the exception of asignificant decrease in the fraction of [14C]TMAcatabolized, total substrate catabolism was simi-lar in sediment with or without BES (Tables 1and 2). Total methane production increased inmolybdate-amended sediments (5.1 nmol/cm3per h) relative to unamended controls (0.92nmol/cm3 per h) and decreased upon addition ofBES (0.75 nmol/cm3 per h). During the course ofthe inhibition experiments, total methane pro-duction was decreased by only 18% in the pres-ence of 27 mM BES. Subsequent experimentsindicated that longer incubation periods (up to48 h) were necessary to achieve complete inhibi-tion of methanogenesis by BES.The addition to ANS of substrates at 1 mM

concentrations resulted in increased methaneproduction during a 48-h incubation (Table 3).The greatest overall stimulation resulted fromamendments with TMA, followed by MA andthen acetate. Incubation with methanol resultedin no notable stimulation (Table 3). Stimulationof methane production by the above-mentionedsubstrates was measurable within 6 h of incuba-tion. Similar results were obtained when BSwere used, though rates of methane productionwere lower.

DISCUSSIONIn Lowes Cove surface sediments, as in other

marine sediments, sulfate reduction greatly ex-ceeded methanogenesis. Rates of sulfate reduc-tion for the 0- to 10-cm depth interval in LowesCove during summer months were typically 2orders of magnitude higher than methane pro-duction (>100 nmol of sulfate reduced per cm3per day versus <7 nmol of CH4 produced per

TABLE 2. Effects of selective inhibitors onsubstrate metabolism in ANS slurries

% Added

Substrate Inhibitor substrate 14CH'14CO2metabo- ratiolizeda

Acetate Na2MoO4 (20 mM) 1.0 0.4TMA 91.7 2.8MA 83.2 2.1Methanol 71.8 1.8

Acetate BES (27 mM) 66.5TMA 6.0 0.2MA 48.8 <0.01Methanol 75.4

a Fraction of label recovered as 14CH4 + 14CO2 after6 h.

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TABLE 3. Effect of substrate additions on rate ofmethanogenesis in ANS'

Substrate Rate 48 h before Rate 48 h afteraddition addition

None 1.3 (0.5) 2.0 (2.9) 53.8TMA 1.1 (0.3) 45.7 (16.6) 4,054.5MA 0.7 (0.5) 8.9 (5.8) 1,171.4Methanol 1.6 (0.5) 1.4 (0.6) -12.5Acetate 1.1 (0.3) 2.7 (0.6) 145.5

a Final concentration of all substrates was 1 mM.Rates are expressed as nanomoles of methane pro-duced per cubic centimeter of sediment per hour.Values represent the mean of three samples; standarderrors are in parentheses.

cm3 per day in BS). The predominance of sulfatereduction was evident from the almost completeoxidation of [2-14C]acetate to 14CO2 (Fig. 1;Table 1). Inhibition of sulfate reduction in ANSby molybdate resulted in increased formation of14CH4 from [2-14C]acetate; however, acetatewas not a significant methane precursor. Similarobservations have been reported for intertidaland subtidal coastal sediments as well as saltmarsh soils (2, 7, 9, 14, 18, 20, 22).Methanol was also an unimportant substrate

for methanogenesis in surface sediments ofLowes Cove. Though some 14CH4 was formedfrom [14C]methanol, the contribution of metha-nol to total methanogenesis was -2% (Table 1).The lack of significant methanogenesis frommethanol at in situ concentrations or 1 mMadditions may have been due to competition bySRB. Ratios of 14CH4/14CO2 were <0.2 (Table1), significantly lower than the ratio of 3 expect-ed for utilization of methanogens alone (13).Addition of molybdate to ANS resulted in asignificant increase in 14CH4/14CO2 ratios (Table2), which suggested oxidation of methanol bySRB. In view of reports that SRB can usemethanol as an energy source (19), it is notsurprising to find significant methanol oxidationby sulfate reduction in marine sediments.

In freshwater sediments with low sulfate con-centrations, methanol appeared to be metabo-lized primarily by MPB, presumably as a resultof the greater competitive ability of methano-gens in freshwater systems (12). In the onlyother reports of methanol metabolism in marinesediments, Oremland et al. (16) and Oremlandand Polcin (17) indicated that methanogenesiswas stimulated by 10 mM methanol; however,Oremland et al. (16) and Oremland and Polcin(17) also indicated that methanol was apparentlynot used by SRB at similar concentrations.Further studies are needed to establish the con-ditions under which methanol may be a signifi-cant source of methane or substrate for sulfatereduction. Differences among sediments with

respect to sources of methanol, such as pectins,should receive particular attention.Because MA concentrations were not detect-

able, MA presumably accounted for a negligiblefraction of total methane production, eventhough a significant fraction of added ["4C]MAwas converted to 14CH4. Potential MA metabo-lism, like that of methanol, appeared to be theresult of both sulfate reduction and methanogen-esis, as indicated by 14CH4/14CO2 ratios andresults of experiments with molybdate and BES(Tables 1 and 2). Data from Lowes Cove inter-tidal sediments were consistent with reports ofMA metabolism in other intertidal sediments(27). Unlike methanol, however, MA additions(1 mM final concentration) resulted in an imme-diate and marked stimulation of methanogenesis(Table 3), suggesting that populations of MPB inLowes Cove are better adapted to compete forMA than for methanol.Of the varied substrates examined, TMA ac-

counted for the greatest fraction of total meth-ane production (Fig. 2; Table 1). In BS whereorganic matter is relatively low, TMA accountedfor 61% of total production, whereas in ANS,TMA accounted for somewhat less. The differ-ence between these two sediments may haveresulted from a greater availability of H2 in theANS due to the incorporation of significantquantities of A. nodosum organic matter into thesediment.Oremland et al. (16) have noted that TMA

turnover could account for 90% of the methaneproduction in slurries of a salt marsh soil amend-ed with Spartinafoliosa. The greater importanceof TMA in marsh soils than in Lowes Cove maybe the result of greater concentrations and pro-duction of TMA in marshes. Methylated aminesare found in high concentrations in halophytessuch as Spartina and may represent readilyavailable sources of TMA (4, 6, 24; G. M. King,Abstr. Annu. Meet. Am. Soc. Microbiol. 1983,1166, p. 167). Comparisons ofTMA metabolismamong sediments must therefore take into con-sideration variability in TMA sources if differ-ences are to be understood or explained.[14CITMA metabolism in both ANS and BS

resulted in higher "'CO2 formation than expect-ed from uptake by methanogens alone; incuba-tion of sediments with molybdate yielded ratiosmore consistent with methanogenesis (Tables 1and 2). These data suggested that TMA utiliza-tion resulted from both sulfate-reducing andmethanogenic activity. However, it should benoted that TMA metabolism has not been dem-onstrated for cultures of sulfate reducers. SRBhave been reported to metabolize choline, acommon methylated amine (5, 19). Studies withcell-free extracts and cultures demonstrated thatacetate and TMA were formed from choline, but

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METHANE PRECURSORS IN MARINE SEDIMENTS 1853

further metabolism of TMA by sulfate reducershas not been described (5, 19).

In summary, neither acetate, methanol, normethylamine was a significant methane precur-sor in Lowes Cove. Acetate and methanol me-tabolism at in situ concentrations were charac-terized by sulfate reduction, whereas that ofMAappeared to result from both sulfate-reducingand methanogenic activity. However, methaneproduction from MA was insignificant becauseconcentrations were not detectable. TMA utili-zation at in situ concentrations accounted for 35to 61% of total methane production. The fractionof methane derived from TMA appeared todepend in part on sediment organic matter con-centrations. Inhibition experiments with molyb-date and BES suggested that TMA was alsooxidized by a process other than methanogene-sis and that in contrast with previous reports (16,17), competition between sulfate reducers andmethanogens may include methanol and methyl-ated amines as well as H2 and acetate.

ACKNOWLEDGMENTSWe thank G. Walker and S. Romatzick for technical sup-

port.This work was funded by National Science Foundation

(NSF) grant 81-09994 and by the NSF Experimental Programto Stimulate Competitive Research.

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12. Lovley, D. R., and M. J. Klug. 1983. Methanogenesisfrom methanol and methylamines and acetogenesis fromhydrogen and carbon dioxide in the sediments of a eu-trophic lake. Appl. Environ. Microbiol. 45:1310-1315.

13. Mah, R. A., M. R. Smith, J. Ferguson, and S. Zinder.1981. Methanogenesis from H,-CO2, methanol, and ace-tate by Methanosarcina, p. 131-142. In H. Dalton (ed.),Microbial growth on C-1 compounds. Heyden and Sons,Ltd., London.

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