JEFFREY T. GELWICKS,'t J. BRUNO RISATTI,2* AND J. M. HAYES'Biogeochemical Laboratories, Departments of Chemistry and Geological Sciences,Indiana University, Bloomington, Indiana 47405,2 and Microbial Geochemistry
Laboratory, Illinois State Geological Survey, Champaign, Illinois 618202
Received 2 April 1993/Accepted 18 November 1993
The carbon isotope effects associated with synthesis of methane from acetate have been determined forMethanosarcina barkeri 227 and for methanogenic archaea in sediments of Wintergreen Lake, Michigan. At37°C, the 13C isotope effect for the reaction acetate (methyl carbon) -> methane, as measured in replicateexperiments with M. barkeri, was - 21.3%o ± 0.3%o. The isotope effect at the carboxyl portion of acetate wasessentially equal, indicating participation of both positions in the rate-determining step, as expected forreactions catalyzed by carbon monoxide dehydrogenase. A similar isotope effect, - 19.2%c ± 0.3%o, was foundfor this reaction in the natural community (temperature = 20°C). Given these observations, it has beenpossible to model the flow of carbon to methane within lake sediment communities and to account for carbonisotope compositions of evolving methane. Extension of the model allows interpretation of seasonal fluctuationsin 13C contents of methane in other systems.
By consuming products of fermentation, methanogenic ar-chaea (32) play a key role in the anaerobic decomposition oforganic matter (20, 33). Cleavage of acetate,
*CH3COOH*CH4 + CO' (1)
is the major source of methane in near-surface, freshwaterenvironments, commonly accounting for 54 to 73% of methaneproduction in sediments (2, 6, 15, 17, 24, 30) and sludgedigestors (14, 26). Reduction of CO, commonly accounts forthe remaining methane produced in freshwater sediments (27).On average, carbon isotope fractionations associated with
the production of methane in freshwater environments aresmaller than those encountered in marine environments (28).Differences in isotope effects associated with production ofmethane from CO, and from acetate have been invoked toexplain this difference (28). Krzycki et al. (16) report theisotope effect (£) associated with aceticlastic methanogenesisto be - 21.2%o at 37°C, a value significantly smaller than the-34 to -46%o observed for reduction of CO2 (3, 8-10, 16).To further extend the study of the isotope effects associated
with aceticlastic methanogenesis, we have grown M. barkeri 227on acetate in closed systems and have measured at variousstages of growth the carbon isotope composition of the acetatesubstrate, the methyl carbon of the acetate, and the methaneproduced. In addition, to determine the isotope fractionationassociated with aceticlastic methanogenesis in a natural micro-bial community, we have amended freshwater lake sedimentswith acetate of known isotope composition and have moni-tored isotope compositions of the sedimentary acetate andmethane.
* Corresponding author. Mailing address: Microbial GeochemistryLaboratory, Illinois State Geologic Survey, 615 E. Peabody Dr.,Champaign, IL 61820. Phone: (217) 333-5103. Fax: (217) 244-2785.
t Present address: Merck Chemical Manufacturing Division, Merckand Co. Inc., Albany, GA 31708.
MATERIALS AND METHODS
Microbiology. Stock cultures of M. barkeri 227, maintainedin the Microbial Geochemistry Laboratory of the Illinois StateGeological Survey, were grown in the following medium(values in grams per liter): KH2P04, 0.24; K2HPO4, 0.24;(NH4)2SO4, 0.24; NaCl, 0.48; FeSO4 7H2O, 0.002;NiCl, * 6H20, 0.0005; (Na)2Se04, 0.0019; MgSO4 * 7H2O, 0.1;CaCl, * 2H2), 0.0064; resazurin, 0.001; sodium acetate, 8.3;L-cysteine-HCI, 0.5; Na2S - 9H2O, 0.5; and NaHCO3, 4.0.Anaerobic medium was prepared by the Hungate technique asmodified by Bryant and Robinson (5). The medium, withoutL-cysteine-HCl, Na2S, or NaHCO3, was boiled under a nitro-gen atmosphere until reduced, and then cysteine and sodiumsulfide were added. The reduced medium was cooled undernitrogen and brought into an anaerobic hood (atmosphere of95% N2 and 5% H2), and 83-ml aliquots were dispensed into160-ml hypovials (Wheaton Glass Co., Millville, N.J.). Theheadspace gases in the sealed hypovials were exchanged threetimes with N2 to a final pressure of 5 lb/in2/g with a gassingmanifold described by Balch and Wolfe (1). Anaerobic sodiumbicarbonate buffer was added to each vial by syringe to aconcentration of 4 g/liter, and the vials were autoclaved at 15-lb(ca. 6.8-kg) pressure for 20 min. The initial pH of the mediumwas 6.9. After autoclaving, the vials were inoculated with 5 mlof M. barkeri 227 culture, which had been grown on medium asdescribed above. Cultures were incubated at 37°C withoutshaking. Growth was monitored by withdrawing 100 [lI ofheadspace gases and measuring methane concentration by gaschromatography. Experiments were performed in duplicate,and anaerobic and sterile techniques were used throughout.
Cultures were harvested at times ranging from 0 to 15 days;headspace gases were transferred into evacuated serum bot-tles, and cells were then removed from the medium byfiltration (Millipore HVLP 04700 filter) under vacuum. Cell-free culture medium containing the residual acetate was keptat -10°C until analyzed. The stoppers of the gas sample vialswere coated with Apiezon W (Apiezon Products Ltd., London,England) to prevent gas loss; vials were kept at room temper-ature until analysis.To prepare a zero-time (to) blank, cells were removed
immediately after inoculation of vials. The isotope composi-tion of the acetate in this sample was considered to be that ofthe initial acetate (i.e., at to) in each vial.
Natural sediments. Surficial sediments were collected fromwithin the 6-m depth contour of Wintergreen Lake, a shallow(maximum depth = 6.5 m), hypereutrophic lake located withinthe W. K. Kellogg Bird Sanctuary, Hickory Corners, Mich.Seasonal changes in the input of organic matter (22) and therate of methanogenesis (17) have been described previously.Sediments were collected with an Eckman dredge, transferredto Mason jars, and stored at 4°C until the experiment wasperformed.
Five milliliters of oxygen-free, 1.0 M sodium acetate and 50ml of Wintergreen Lake sediment were added anaerobically toeach of nine 125-ml serum bottles. The carbon isotope com-position of the sodium acetate was - 17.4%o ± 0.2%o. Vialswere capped with butyl rubber stoppers and incubated at 20°Cwith shaking. Individual bottles were harvested at times rang-ing from 1 to 9 days by collecting headspace gases, centrifugingthe vial, and decanting the liquid phase. Liquids were storedfrozen until they were analyzed. The to vial was harvestedimmediately after addition of acetate and sediment. Theamount and isotopic composition of methane in the to vial wasdetermined and taken into account as a blank in the analysesof all subsequent samples; the isotope composition of acetatein the to vial was taken as representative of the initial acetate inall vials. The isotope composition of total acetate in Winter-green Lake sediments was determined to be -18.1%o +0.1%o.
Chemical and isotopic analyses. Acetate was analyzed iso-topically by the procedure described by Gelwicks and Hayes(11). Briefly, liquid phases were adjusted to pH 10 by additionof NaOH and evaporated to dryness. Aliquots of residualsolids were mixed with oxalic acid dihydrate and heated undervacuum until the mixture melted (102°C). The ensuing acid-base reaction produced sodium oxalate and acetic acid, thisbeing purified by gas chromatography (glass column; insidediameter, 5 m by 4 mm; packed with 80/100-mesh Porapak Q;helium carrier gas flowing at 20 to 25 ml/min) and combustedon line (850°C) in a quartz tube furnace packed with cupricoxide. The resultant CO2 was cryogenically distilled and col-lected for isotopic analysis.To determine the isotope composition of the methyl carbon
of acetate, the chromatographically purified acetic acid wastrapped on NaOH pellets and pyrolyzed at 500°C in order toproduce CH4 from the methyl position (21, 23). Methane wascombusted at 850°C, and the resultant CO2 was cryogenicallypurified. Completeness of combustion and accuracy of isotopicresults were tested by analysis of methane standards.
Analyses of a sodium acetate standard (8 = 29.5%o +0.1%o) by this procedure yielded an average result of - 29.3%o± 0.3%o (n = 8) for samples ranging in size from 1 to 100,umol of acetate. In the present application, sample sizesranged from 3 to 81 ,umol of CO2. Pyrolytic yields of CH4ranged from 93 to 98%, and the isotope composition wasindependent of yield. Replicate analyses yielded a standarddeviation of 0.4%o (n = 4).
Bacterial methane was collected on Porapak Q at - 196°Cafter removal of water at - 131°C and carbon dioxide at- 196°C. No carbon-bearing compounds other than methaneand carbon dioxide were present. Analyses of a methanestandard (8 = - 47.70%o ± 0.15%o) by this procedure yieldedan average result of - 47.77%o ± 0.14%o (n = 4).Carbon isotope ratios were measured with Nuclide 6-60 and
Finnigan MAT Delta E isotope-ratio mass spectrometers.Results are reported in the delta notation:
TABLE 1. Fractional yields (based on methyl carbon) and 13Cisotope compositions of substrates and product from pure
culture experiments
Isotope composition (%o)afbExpt Total Methyl Methane
a Versus PDB standard.b Calculated from equation 7.
813CPDB = [(RSamPIe - RPDB)/RPDB] X 103 (2)
where R = 13C/12C and RPDB = 0.0112372 (PDB refers toPedee belemnite standard). Units for 8 and for measuredisotope effects are parts per thousand, termed permil, andassigned the symbol %o.
Calculations. The equations presented by Mariotti et al. (18)have been used to calculate - from fractional yields and isotopecompositions of substrates and products. Specifically, calcula-tion of s can be based on the isotope composition of theresidual reactant:
8af= bai + E[ln(1 - f)] (3)wheref is the fractional yield based on consumption of acetate(O < f < 1), baf is the isotope composition of methyl carbon inacetate at any f, and bai is the initial isotope composition ofmethyl carbon (f = 0). An independent determination of - canbe based on measured isotope compositions of the product:
8mf = 8ai - s(1 - f)[ln(1 - f)]f (4)where 8mf is the isotope composition of pooled methane at anyf. Linear regression yields s as the slope of best-fit lines when8af is plotted as a function of ln(1-) and when bmf is plottedas a function (1 - )[ln(1 - )]/f.From mass balance, isotope compositions will be related as
8ai = (nminai)8mf+ (nfa/nai)8af (5)where n's designate molar amounts and subscripts are asdefined above. Since f = nnm/nai and nmf + naf = nai,
8ai -2 fI8mf + (1 - h8'af (6)
Rearrangement yields an expression forf in terms of measuredisotope compositions:
f = (i - 8af)/(8mf - 8af) (7)
RESULTS AND DISCUSSIONGrowth of M. barkeri. Results are summarized in Tables 1
and 2. The duplicate experiments yielded slightly differentresults, both in terms of calculated E values and in terms ofprecision. In experiment 1, in which the range off values wassmall, the calculated uncertainties in s were also small. Inexperiment 2, both the range of f values and the calculateduncertainties in E were larger. It could be argued either thatthe more precise result should be preferred or that the
a Versus PDB standard.b Calculated from equation 7.
experiment in which the reaction was monitored well beyondany low-conversion artifacts was superior. We favor the valuesobtained in experiment 2 because precision is still excellent bycomparison with most measurements of microbial isotopeeffects and because results agree with the value obtained in aprior measurement (16). Results of both experiments (Table 2)suggest but do not prove that the isotope effect associated withconsumption of methyl carbon is slightly larger than thatassociated with production of methane. This could indicateeither that some branch point between assimilation of methylcarbon and release of methane drew off a "3C-depleted streamto an alternate fate such as cellular biosynthesis or that theisotope composition of methane was being affected by somedownstream reaction that consumed 12CH4 in preference to'3CH4. Although this difference between the methyl isotopeeffect and the methane isotope effect is not statistically signif-icant, the possibility that such phenomena may be reliablydetectable in more precise experiments should be considered.The acetyl-CoA pathway. Results of both experiments indi-
cate that the fractionation of '3C in total acetate is significantlygreater than that at methyl carbon. If the reaction rate weresensitive only to isotopic substitution at the methyl carbon, theisotope effect determined by analysis of total acetate would behalf as large as that measured by analysis of the methyl carbon.The fact that it is not smaller but is, in fact, marginally largerindicates sensitivity to isotopic substitution at both positionsand, therefore, involvement of both positions in the rate-determining step of the overall reaction. This is consistent withattribution of the isotope effects to processes catalyzed bycarbon monoxide dehydrogenase (CODH). Cleavage of ace-tate by methanogenic archaea occurs via the acetyl coenzymeA (acetyl-CoA) pathway, in which acetate is converted toacetyl-CoA for use either in anabolic reactions or in theproduction of methane (7, 13, 34, 35). The central reaction inthe pathway involves CODH, which catalyzes cleavage ofacetyl-CoA, the oxidation of the carbonyl group to CO2, andtransfer of the acetate methyl group to an acceptor to ulti-mately be transferred to HS-CoM (7, 13). Isotopic fraction-ations accompanying methane production from methanol,CO2, and acetate (16) indicate that isotopic fractionationoccurs prior to the formation of methyl-CoM. The presentresults are most simply explained if isotope effects at bothcarbon positions of acetate are associated with the activity ofCODH.
Isotopic discrimination has also been observed during pro-duction of acetate by Acetobacterium woodii (12), which uti-lizes the acetyl-CoA pathway analogously, but in reverse, to M.barkeri (13, 34). Isotope effects at the methyl and carboxylpositions in acetate produced from CO2 and H2 are equal(-58.6%o ± 0.7%o [12]). In A. woodii, CODH also mediates
exchange reactions between the carboxyl carbon of acetate andCO2 (31).
Utilization of acetate in a natural community of methano-gens. It is shown in Table 3 and Fig. 1 that similar isotopeeffects are associated with consumption of acetate and produc-tion of methane by a natural community. In these experiments,no attempt was made to inhibit production of methane oracetate from dissolved CO2 by methanogens or acetogens. InWintergreen Lake, the source of these sediments, approxi-
1-
a -20 .5-a.0
u1
u -21.0 -Vto
A
£ = -19.2 ± 0.2%o
A
.0* 0 .... ..II-0 00 00 00 01 0
-0.07 -0.06 -0.05 -0.04 -0.03 -0.02 -0.0 1 0 0.01
ln(1-f)
-39.8-
-40.0-
-
Q
Vw
-40.2-
-40.4 -
-40.6-
.40.8
-41.0 l,,. I.. ,....,.. I..,.I . I.. ... I. .I
- 1 -0.995 -0.99 -0.985 -0.98 -0.975 -0.97 -0.965
(1-f)ln(1-f)/fFIG. 1. E values from Lake Wintergreen sediment experiments
based on methyl carbon of acetate substrate (equation 3 and columns1 and 3 of Table 3) (A) methane product (equation 4 and columns 2
mately 38% of methane derives from reduction of CO2 (17),and the 3%o difference between the methyl carbon consump-tion and methane production isotope effects may reflect acontribution of "3C-depleted methane from that pathway.The observation that the isotopic fractionation measured
with the pure culture is similar to that measured with thenatural community is somewhat surprising. Kinetic isotopeeffects associated with CO2 reduction as measured in purecultures vary widely between species (2, 8-10, 16). A naturalcommunity of methanogens using CO2 would be expected toproduce methane having an isotope composition equal to theweighted average of the inputs from each species, not neces-sarily equal to that of one species in particular. The similarityin s observed here between the pure culture and the naturalcommunity of aceticlastic methanogens may indicate (i) thatM. barkeri is the only aceticlastic methanogen in these sedi-ments or (ii) that all aceticlastic methanogens in these sampleshave similar isotope effects. Species of aceticlastic methano-genic archaea other than M. barkeri have been isolated in purecultures (29); their presence in Wintergreen Lake sedimentsshould be considered. However, if the acetyl-CoA pathway andCODH are employed by all aceticlastic methanogens, a similarisotope effect should accompany methanogenesis by differentaceticlastic methanogens.Methane produced in freshwater environments. Even in
environments in which aceticlastic methanogenesis is the dom-inant source of methane, the isotope effect measured in thisstudy will not be the only factor controlling the isotopecomposition of methane. Methane is generated in freshwaterenvironments both from cleavage of acetate and from reduc-tion of CO2. The isotopic composition of methane leavingthe sediment will reflect that combination and can be calcu-lated as
(Pm = (Pma + (Pmc (8)
(Pm8m = 'Pma8ma + (Pmc8mc (9)where p terms represent carbon fluxes and 8 terms representisotope compositions. The subscripts m, ma, and mc representtotal methane, methane from aceticlastic pathways, and meth-ane from reduction of C02, respectively. Dividing equation 9by Pm, we obtain
am = ((PmalPm)ma + ((Pmcl(m)8mc (10)and defining Pma/lPm = Xa = the fraction of methane producedfrom acetate and Xc = piPmc/pm = the fraction of methaneproduced from CO2 and then substituting these terms intoequation 10, we can write
am Xabma + Xcbmc (11)where the X values are the mole fractions of methane pro-duced by cleavage of acetate and by reduction of CO2,respectively (Xa = 1 - Xc), and 8ma and 8mc are the isotopiccompositions of methane produced from acetate and fromCO2. Values for 6mc and 6ma cannot be measured directly butcan be determined from known isotope effects and isotopiccompositions of substrates:
reduction of C02: 6mc = 6c + 8mc (12)
cleavage of acetate: bma = 6a + Ema (13)
where 8's represent carbon isotope compositions of sedimen-tary CO2 (c) and acetate (a) and £'s represent isotope effectsassociated with methane production from C02 (mc) and fromacetate (ma). In order to model methane production, each ofthese factors must be considered.
TABLE 4. Seasonal variations in 813C of methane and CO2 fromCape Lookout Bight sediments and estimated fractions
of methane derived from Xa or Xc
Date Isotope composition (%c)(mo/day/yr) 813C(methanc) 83C(co2) x, Xa
The sediments of Wintergreen Lake can be considered as anexemplary methanogenic system, and equations 10 to 13 can beused to examine carbon budgets within the system. Specifically,values of Xc and Xa can be obtained if 8m' 6a' 6c, Ema' and 6mcare known. The present work has supplied £ma = - 21.3%o.Measured values of 6m from Lake Wintergreen sedimentsrange from -56 to - 59%o, and analyses of these sedimentshave yielded 6c = - 5%o, and 8a = - 20.4%o. The isotopeeffect associated with the reduction of CO2 (smc) has beendetermined for pure cultures, but values have varied greatlybetween species and do not appear to be representative ofnatural environments. A useful value of Emc for sedimentaryenvironments, however, derives from the work of Whiticar etal. (28), who showed that the isotope effect accompanyingproduction of CH4 from CO2 in marine sediments amountedto 73%o. If that value is adopted as generally representative ofnatural communities that produce CH4 by reduction of CO2,then 6mC = - 73%o together with the values noted above canbe inserted in equations 11 to 13. Calculation of equation 11then yields 0.52 - X - 0.61 and 0.48 2 Xc 2 0.39, theindicated range corresponding to -59 am6< -56%o. Therange obtained for Xc can be compared with the results oflabeling experiments that indicated Xc = 0.38 (17).
Application of model to methane production in Cape Look-out Bight sediments. Cape Lookout Bight (North Carolina) isa marine environment characterized by high influxes of organicmatter and by high sedimentation rates. Decomposition oforganic matter in these sediments is dominated by a zone ofsulfate reduction overlying a zone of methanogenesis. Seasonalvariations in the carbon isotope composition of methaneproduced in these sediments have been attributed to seasonalchanges in the major pathways of methane production (19).Estimates ofXc and Xa can be calculated from data reported byMartens et al. (19); results of these calculations are shown inTable 4. Blair et al. (4) reported 6a for these sediments as- 26.4%o (sample collected 14 June 1984); although this isonly a single measurenrent, and temporal variations in 6a areexpected, this value was used to produce seasonal estimates ofXc.Model calculations indicate that the relative amount of
methane produced from reduction of CO2 varied throughout
13C EFFECTS ASSOCIATED WITH ACETICLASTIC METHANOGENESIS 471
the year, ranging from 27% in August 1983 to 60% in May1984. The depth distributions of the zones of sulfate reductionand methanogenesis vary seasonally and are controlled byvariations in organic matter input and temperatures (19, 25).High values of X, correlate with times (generally February toMay) during which the zone of sulfate reduction extendeddeepest into the sediment and sediment temperature and rateof input of organic matter were low. Sulfate-reducing bacteriawere the major sink for acetate (25), and methanogenesisoccurred mainly from reduction of CO2 during those months.High values of Xa correlate with times in which the zone ofsulfate reduction is thin and rates of organic matter input arehigh. The high organic loading of the sediment likely results inhigh rates of acetate production. The activity of sulfate-reducing bacteria becomes limited by available sulfate andacetate thus becomes available for methanogenesis. As alsoobserved by Risatti (24), temporal changes in the availability ofacetate for methane production are due to available organiccarbon inputs and result in the variations observed in thecarbon isotope composition of methane produced in sedi-ments. These calculations are consistent with observations (19)that seasonal variations in Bm can be due to fluctuations in themajor pathways of methane production. However, temporalvariations in the isotopic composition of sedimentary acetatewill also influence the isotopic composition of sedimentarymethane and should be examined.
ACKNOWLEDGMENTS
This work was supported in part by the National Aeronautics andSpace Administration (NGR 15-003-118) and by the NASA PlanetaryBiology Internship program (J.T.G.).We thank M. J. Klug for assistance and use of laboratory and
sampling equipment and W. Jack Jones for assistance with M. barkericultures in the early stages of this work.
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