STABLE ISOTOPE TRACING OF ANAEROBIC METHANEOXIDATION IN THE GASSY SEDIMENTS OF ECKERNFORDE BAY,

GERMAN BALTIC SEA

CHRISTOPHER S. MARTENS, DANIEL B. ALBERT, AND M. J. ALPERINDepartment of Marine Sciences,

University of North Carolina at Chapel Hill,Chapel Hill, North Carolina 27599-3300

ABSTRACT. Methane concentrations in the pore waters of Eckernforde Bay inthe German Baltic Sea generally reach gas bubble saturation values within theupper meter of the sediment column. The depth at which saturation occurs iscontrolled by a balance between rates of methane production, consumption(oxidation), and transport. The relative importance of anaerobic methane oxida-tion (AMO) in controlling dissolved and gas bubble methane distributions in thebay’s sediments is indirectly revealed through methane concentration versusdepth profiles, depth variations in the stable C and H isotope composition ofmethane, and the C isotope composition of total dissolved inorganic carbon(�CO2). Direct radiotracer measurements indicate that AMO rates of over 15 mMyr�1 are focused at the base of the sulfate reduction zone. Diagenetic equationsthat describe the depth distributions of the �13C and �D values of methanereproduce isotopic shifts observed throughout the methane oxidation zone andare best fit with kinetic isotope fractionation factors of 1.012 � 0.001 and 1.120 �0.020 respectively.

INTRODUCTION

In late 1972, the senior author of this paper was introduced to the interesting topic ofgassy sediments while working in the laboratory of Professor Robert A. Berner at YaleUniversity. We were seeking to measure concentration distributions of dissolved N2 andAr in the pore waters of anoxic sediments in Long Island Sound to study possiblechemical reactions involving either N2 production or reduction. While making thesemeasurements using a gas partitioner and modifications (Martens, 1974) of techniquesdeveloped by Reeburgh (1968), an unidentified peak appeared at the end of some, butnot all, of the lengthy chromatographs. ‘‘Chromatogram-watching’’ until the paper runsout is a favorite pastime of wannabe treasure hunters turned chemists! Curiosity won out,and it was soon determined through the assistance of Danny Rye, who had a real gaschromatograph for studies of the geochemistry of fluid inclusions, that the unidentifiedgas was methane. Even more fascinating was the fact that variations in the concentrationof dissolved methane in the sediments of Long Island Sound appeared to be inverselycorrelated to the concentration of dissolved sulfate in the pore waters. Dissolved sulfatewas being measured as part of our nutrient regeneration study (see Martens et al., 1978).The field data demonstrated that dissolved methane occurred at shallower depths inanoxic sediments featuring almost complete sulfate depletion. To further investigate thisphenomenon, we proceeded to undertake balmy, wintertime sampling visits to varioussmall harbors, some of which remain famous for their impact on sulfide-challengedindividuals as well as revelations concerning authigenic phosphate mineral formation(Martens et al., 1978; Ruttenberg and Berner, 1993). The full data set from a variety ofsites in Long Island Sound revealed that ‘‘high methane concentrations do not occurunless sulfate concentrations have been appreciably lowered.’’ Following a harrowingreview experience during which existing but incorrect paradigms based primarily onpreceding laboratory studies were asserted to invalidate our conclusions based on thefield data, our initial contribution to this topic area including the quote above, appeared(Martens and Berner, 1974). Four alternative hypotheses were advanced in this paper,including ‘‘that methane is produced only in the absence of dissolved sulfate; however,on diffusing upward, is then consumed by sulfate reducers.’’ The development of this

[AMERICAN JOURNAL OF SCIENCE, VOL. 299, SEPT., OCT., NOV., 1999, P. 589–610]

589

alternative into a full-blown hypothesis deserving extensive attention and researchresulted from the work of many people led by investigators such as Barnes and Goldberg(1976), Reeburgh (1976), Reeburgh and Heggie (1977), as well as ourselves (Martens andBerner, 1977).

Methane Production in Organic-Rich Marine SedimentsMethane production occurring in organic-rich, marine sediments is controlled by a

complex set of coupled biogeochemical processes associated with the bacterial reminer-alization of labile organic matter. Organic matter remineralization in sediments proceedsthrough a sequence of reactions involving a variety of oxidants. If O2 is present, aerobicdecomposition is generally the major pathway. Upon depletion of dissolved O2, organicmatter degradation shifts to reduction of nitrate and reactive metal oxides (MnO2 andFe2O3. Following removal of the oxidants organic matter remineralization proceedsthrough sulfate reduction. The final reaction in the series, methane production, generallyoccurs after the sulfate pool has been exhausted (Martens and Berner, 1974). Exceptionsinclude production of methane from methanol and methylated amines (Oremland,Marsh, and Polcin, 1982; Lee and Olson, 1984).

Gassy sediments are most commonly found in shallow, productive environmentsthat receive a large flux of reactive organic matter (Mechalas, 1974). At these sites,dissolved oxygen is depleted within several millimeters of the sediment-water interface(Revsbech and others, 1980; Grundmanis and Murray, 1982; Reimers, Jahnke, andMcCorkle, 1992). Nitrate reduction is thought to be a minor oxidant for organic carbondue to its low concentration in the overlying seawater (Murray, Grundmanis, andSmethie, 1978; Reeburgh, 1983). Organic matter remineralization coupled to metaloxide reduction appears to be limited to surficial sediments that are rapidly mixed bybioturbation (Canfield, Thamdrup, and Hansen, 1993) or physical processes (Aller,Mackin, and Cox, 1986).

In sediments prone to gas production, sulfate reduction and methane productionare likely to be the dominant decomposition reactions (Martens and Klump, 1984). Theoccurence of these and most other biogeochemical processes in coastal sediments isultimately controlled by the flux of reactive organic matter; however, the rates and depthdistributions of these reactions vary dramatically in response to seasonal variations intemperature ( Jørgensen, 1977; Martens and Klump, 1984; Crill and Martens, 1983,1987). Following its production, the distribution of methane in sediments is furthermodified by consumption (oxidation), gas bubble formation, and transport processeswhich may also exhibit pronounced seasonality in their absolute rates and relativeimportance.

Anaerobic Methane Oxidation at the Base of the Sulfate Reduction ZoneA recent review of present knowledge concerning anaerobic methane oxidation

(AMO) has been presented by Hoehler and Alperin (1996), and the reader is referred tothat paper for a comprehensive literature review and summary. Environmental studies ofAMO have consisted of at least five independent approaches including diageneticmodels, thermodynamic calculations, radiotracer experiments, sediment time seriesincubations, and stable isotope measurements. Of particular interest here are the studiesof Reeburgh (1980) in which rate distributions of AMO were measured in the anoxicsediments of Skan Bay, Alaska, and the following study of Alperin, Reeburgh, andWhiticar (1988) which documented carbon and hydrogen stable isotope fractionationresulting from AMO at that site.

More than a decade following Zehnder and Brock’s (1979) suggestion that methano-gens themselves might be capable of net methane oxidation given appropriate environ-mental conditions, Hoehler and others (1994) demonstrated that control of pore waterdissolved H2 concentrations by sulfate reducers could provide the lowered concentra-

Christopher S. Martens and others—Stable isotope tracing590

tions necessary to support net methane oxidation by methanogens in the anoxicsediments of Cape Lookout Bight, North Carolina. This hypothesized methanogen-sulfate reducer consortium explanation appears to be a biochemically and thermodynami-cally feasible (Hoehler and others, 1998) mechanism which can explain net methaneoxidation in other anoxic marine sediment and water column environments.

Study ObjectiveThe purpose of this paper is to present a relatively complete new set of pore water

concentration, biogeochemical rate, and light stable isotopic data from a gassy coastalsediment which can be combined to further elucidate the role of AMO and otherprocesses in controlling methane concentrations and net transport in organic-richenvironments. The opportunity for this research came through the Coastal BenthicBoundary Layer (CBBL) interdisciplinary research program (Richardson, 1994) led byscientists from the Naval Research Laboratory, Stennis Space Center, and the Forshung-sanstalt der Bundeswehr fur Wasserschall und Geophysik (FWG) of Kiel, Germany.Field studies associated with this program were focused on integrated acoustical,sediment geotechnical, oceanographic, and biogeochemical process measurements inEckernforde Bay, a small basin in the Kiel Bight of the German Baltic, containingextremely gassy sediments.

The study site: Eckernforde Bay.—Eckernforde Bay is an elongate inlet extendingsouthwestward from the Kiel Bight of the western Baltic Sea (fig. 1). The Bay’s centralbasin reaches depths of 28 m and is underlain by fine-grained, anoxic mud which canexceed a thickness of 7 m. A summary description of the physical processes controllingwater column salinity, density structure, and sedimentation processes in EckernfordeBay is provided by Friedrichs and Wright (1994). Erosional currents rarely affect areas at

Fig. 1. Location of the Eckernforde Bay study site, western Baltic Sea. Results presented in this paper wereobtained at the NRL station.

of anaerobic methane oxidation in the gassy sediments 591

water depths greater than 20 m, and sediment mud content generally increases withbasin depth (Werner, 1987; Werner and others, 1987). Measured sediment accumulationrates in Eckernforde Bay range from 0.3 to 1.1 cm yr�1 (Nittrouer and others, 1998) withan average value of 0.6 cm yr�1 at the NRL site utilized in this study. Bioturbation hasonly been observed to influence sediment properties within the upper few millimeters ofthe sediment column at the NRL site.

Earlier investigations of methane distribution and stable isotopic composition in thepore waters of nearby sites in Eckernforde Bay are described by Whiticar and colleagues.Whiticar (1982) found saturation concentrations of methane at depths greater than 4.5 malthough possible gas losses during sediment core retrieval and storage were noted.Whiticar and Werner (1981) reported C and H stable isotopic values for methane fromEckernforde Bay which were later included by Whiticar, Faber, and Schoell (1986) in aBest Paper Award-winning study of the use of stable isotopic values to distinguishmethanogenic pathways in marine and freshwater environments.

METHODS

All pore water concentration, stable isotope, and biogeochemical rate data wereobtained from samples collected during two cruises occurring during May, 1993 andJuly, 1994. Sediment cores and pore water membrane equilibration (ME) samples werecollected by divers supplied by the German Navy or through the use of large-diametergravity cores collected by the officers and crew of the German ship PLANET operatedby the FWG.

Pore water concentration measurements.—Pore water samples for sulfate and chloridewere collected from gravity cores using syringes fitted with cylindrical porous plastic tipsinserted directly through pre-drilled and taped holes in the sides of core liners. Afterinsertion, the syringe plungers were cocked and left until a few milliliters of porewaterwere collected. After filtration through a syringe filter (0.45µ) a 1 ml aliquot was put in avial for later sulfate analysis. This sample was acidified and bubbled to remove sulfide toprevent it from oxidizing and adding to the measured sulfate concentration. A separatealiquot was saved for chloride analysis.

Both analyses were done using a Dionex 2010i ion chromatograph equipped withan AS4A column. The eluant used was 2 mM in both NaHCO3 and Na2CO3. Samplesand standards were diluted 1:100 with eluant prior to analysis to eliminate the water dipthat otherwise interferes with Cl� analysis. For more accurate determination of lowconcentrations of sulfate a 1:10 sample dilution was used, and the Cl� removed byforcing samples and standards through Ag� form cation exchange cartridges (Dionex) atthe time of injection. This prevented column overload due to the high Cl� concentra-tions at this low dilution. For these samples Cl� was determined separately at the higherdilution.

�CO2 and methane analyses were performed on the ship using a Carle GasChromatograph (Hach Equipment Co.) equipped with both thermal conductivity (forCO2) and flame ionization (for methane) detectors. Both analyses were performed byheadspace equilibration techniques.

For �CO2 analyses 1 ml porewater samples, or sodium carbonate standards, wereinjected into a 10 ml syringe with a three-way stopcock valve followed by 1 ml 10 percentHCl. The valve was closed quickly, and a vacuum was pulled on the sample whileshaking. The valve was then cracked open, and several milliliters of air was pulled infollowed by more shaking for headspace equilibration. The final headspace was made upto 10 ml prior to GC analysis.

Total dissolved and gas bubble methane concentrations were measured on whole-sediment aliquots obtained by sediment subcorers consisting of cut-off 60 ml plastic

Christopher S. Martens and others—Stable isotope tracing592

syringes. Within a few minutes of core retrieval, the sediments were sampled with thesesyringes through the taped holes along the core. Each sample was expelled into a taredjar of known volume with a gas-tight lid. The samples were allowed to warm to ambienttemperature and degas, aided by shaking. The jars were then pierced using a septum-sealed jar piercer (Alltech). To establish accurate headspace volume at atmosphericpressure the headspace pressure built up due to degassing was allowed to displace theplunger of a 60 ml all-glass syringe. This plunger was displaceable with almost noresistance when lubricated with water. The displacement was measured in both thebarrel-pointing-up and barrel-pointing-down positions (to compensate for the weight ofthe glass plunger), and the average reading was added to the headspace volume in thejars for determination of methane content of the samples.

Samples for both �CO2 and methane analyses were injected into the GC by passingthem through a small tube filled with Drierite and filling a fixed volume sample loop.Standards for �CO2 were carbonate solutions carried through the same headspaceequilibration steps. For methane, calibration gasses were premixed methane in air atseveral concentrations (Scott Specialty Gases). Porewater volumes in the bulk sedimentsamples were calculated from sediment weight and porosity. Concentrations werecalculated assuming all methane and �CO2 were dissolved in the porewater (no gasphase). The precision of replicated concentration measurements on a single sample was�3 percent for both �CO2 and methane. Duplicate samples were not analyzed for agiven core.

Stable carbon and hydrogen isotope measurements.—Stable isotope samples were purifiedcryogenically using vacuum line technology. Samples for �13CO2 analysis were acidifiedin the storage vial and bubbled with helium to remove the CO2. The gas stream passedthrough a water trap submerged in pentane/pentane ice slush, and the CO2 was frozenout in a liquid nitrogen trap. After trapping, the helium flow was stopped, and the heliumpulled off under vacuum. The evacuated system was then closed, and the CO2 trans-ferred by warming the trap and retrapping the gas in a closed-end glass tube submergedin liquid nitrogen. After trapping this tube was cut off and sealed with a torch. The CO2samples were transferred to the mass spectrometer in these sealed tubes.

Methane stable isotope samples were also stripped from their vials with flowinghelium. After water removal, the helium stream passed through a furnace where themethane was combusted by passing through a bed of cupric oxide at 800°C. The streamthen passed through a water trap to remove the combustion water and the CO2 washandled as described above. The combustion water was then also transferred cryogeni-cally to a separate glass tube containing ‘‘Indiana zinc,’’ a proprietary, zinc-basedreactant ( John Hayes, Woods Hole Oceanographic Institute). After sealing, these tubeswere heated at 500°C for 30 minutes during which the catalyst quantitatively convertedthe water to hydrogen while scavenging the oxygen produced. The hydrogen gasevolved from the methane was transferred to the mass spectrometer in these flame-sealed tubes.

Stable C and H isotope measurements were made utilizing Finnigan MAT 252Isotope Ratio Mass Spectrometers (IRMS). Samples were collected at the time of �CO2and methane concentration measurements and stored under refrigeration for about amonth before analysis. For �13CO2, 1 ml filtered porewater samples were stored in smallcrimp-sealed serum vials. Methane for �13C analysis was collected by filling a largesyringe with headspace gas from the sealed jars used for headspace analysis and using itto fill an inverted, water-filled serum vial that was held submerged in a bucket of water.After filling, the stopper was put in place while the vial was still under water, then it wasremoved, and the crimp seal applied. ‘‘Delta’’ notation was used for describing carbon

of anaerobic methane oxidation in the gassy sediments 593

and hydrogen stable isotope ratios using the equation:

� (‰) � 3 R(sample)

R(standard)� 14 1000 (1)

where R is the ratio of heavy to light isotope in the sample or the reference standard, andheavier (more positive) values have more of the heavier isotopes (13C or deuterium).

Biogeochemical rate measurements.—Anaerobic methane oxidation (AMO) rates weredetermined by radiotracer techniques using 14C labelled methane as described previ-ously by Hoehler and others (1994). Briefly, labelled methane was injected into 3 mlglass syringe cores taken horizontally through the walls of core barrels. These wereincubated for about 48 hrs at the in situ temperature. The incubation was terminated byexpelling the syringe cores into 5 ml 1 M NaOH solution in serum vials which were thenstoppered and crimp-sealed. The radioactive methane in the sample vials was strippedwith flowing air, which carried it through a furnace where it was combusted over a bed ofcopper oxide at 800°C. The labelled CO2 produced was trapped in a phenethylamine-containing scintillation cocktail and quantified to determine overall recoveries. Follow-ing stripping of the methane the vials were acidified, and labelled CO2 that had beenproduced during the incubation was stripped and trapped in the same cocktail. Duringthis step the gas stream did not go through the furnace but did pass through a trapcontaining 0.5 M zinc acetate solution at pH 4.0. This trapped hydrogen sulfide releasedby acidification of the sediment but allowed the CO2 to pass through to the phenethyl-amine trap. Trap activity was quantified by liquid scintillation counting for 10 min afterthe stripping was finished. Labelled methane and CO2 were both counted as CO2 in thephenethylamine traps, and no quench corrections were performed because only relativeactivities are important. Rates were determined from the fraction of the labeled methaneoxidized per unit time multiplied by the concentration of methane in the sample.Methane concentration in the samples used for AMO rates was determined by analyzingthe headspace of the vials prior to stripping, rather than assuming in situ concentrations.The precision of replicated AMO rate measurements between samples was �17 percent.

RESULTS

Methane pore water concentration data from the 1994 expedition are summarizedin table 1. Samples collected in 1993 suffered gas losses associated with sample retrievaland storage time prior to analysis at a shore laboratory. However, stable isotopic dataobtained during 1993 appear to be unaffected by the gas losses and are included below.During 1994, samples were collected from cores within hours or less of collection andwere analyzed on board ship. Methane equilibration (ME) sampler ports were observedto ‘‘fizz’’ within tens of minutes following collection as a result of oversaturation resultingfrom their return to the surface from gas-saturated sediments at approx 3.8 atm pressure(28 m depth). The ME concentration data are regarded as underestimates and are notincluded in our tabulated data set.

Concentration data for other pore water constituents including salinity, dissolvedsulfate, and �CO2 are summarized in table 2. Additional data from nearby sites,including pockmarks, can be found in Albert, Martens, and Alperin (1998).

Methane Concentration Versus Depth DistributionsSaturation methane concentrations were observed to begin between approx 30 to

75 cm depth during sampling expeditions in May 1993 and July 1994 (fig. 2). Theseresults agree with the recent measurements of methane concentration versus depthprofiles (Abegg and Anderson, 1997) and the depth distribution of acoustic turbidity(Wever and Fiedler, 1995; Abegg and Anderson, 1997; Anderson and others, 1998) at ornear the NRL study site. In general, acoustic turbidity occurs somewhat deeper than the

Christopher S. Martens and others—Stable isotope tracing594

calculated depth of methane saturation (Abegg and Anderson, 1997). Saturation meth-ane concentrations were generally found at 50 cm depth at the NRL site. However, nogas bubble ebullition was observed, and direct bottom surveys by SCUBA divers, as wellas video camera surveillance, revealed no gas bubble losses from the sedimentsthroughout the winter and summer months of 1993 and 1994.

A prominent feature of methane data from the upper meter of the NRL site is anextreme concave-up concentration versus depth distribution of dissolved methane overthe sulfate reduction depth zone (fig. 3). This distribution is recognized to result fromAMO (Martens and Berner, 1974; Barnes and Goldberg, 1976; Reeburgh, 1976;Reeburgh and Heggie, 1977; Martens and Berner, 1977) and appears to be the primaryprocess preventing a significant upward flux of methane from Eckernforde sediments tothe overlying water column.

Pore Water Advective TransportSalinity versus depth profiles at the NRL site (table 1; fig. 2) as well as previous

observations of freshwater flow at nearby pockmark sites (Whiticar and Werner, 1981)clearly indicate the importance of upward pore water advection (Albert, Martens, andAlperin, 1998) associated with the glacial till layer underlying surficial sediments at adepths ranging from approximately four to over five meters. The latter authors calcu-

TABLE 1

Methane concentrations in sediment porewaters at the NRL site, Eckernforde Bay, July 1994

of anaerobic methane oxidation in the gassy sediments 595

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Christopher S. Martens and others—Stable isotope tracing596

lated an advection rate, �, of 1.0 cm yr�1 from the salinity versus depth profiles using asimple steady-state advection-diffusion equation.

Methane Stable Carbon and Hydrogen Isotope DistributionMethane stable C and H isotope data are summarized in table 3. The data exhibit

extremely smooth and reproducible distributions, with distinct maximum and minimumvalues corresponding to depth changes or to major shifts in pore water chemicalcomposition, including dissolved sulfate depletion and �CO2 concentration increasewith depth. The measured �13C values of �CO2 are reported in table 4.

Fig. 2. Methane concentration and salinity versus depth at the NRL site in 1994. The shaded dots refer todata (table 1) from cores G-2 (dark) and 673 (gray). The dashed line is calculated methane saturation versusdepth.

of anaerobic methane oxidation in the gassy sediments 597

DISCUSSION

Kinetic Model Results for Eckernforde Bay Methane Concentrations and AMO RatesMartens, Albert, and Alperin (1998) have presented a kinetic model derived from

the original of Berner (1980) which is designed to predict biogeochemical processesoccurring in gassy, anoxic sediments dominated by sulfate reduction (SR), methaneproduction (MP), and anaerobic methane oxidation (AMO). The model is composed ofmass conservation equations in which reaction rates are balanced by diffusive andadvective transport. It directly couples biogeochemical zones using error functions thatserve as a toggle to simulate cessation of sulfate reduction, initiation of methaneproduction and oxidation, and production of gaseous methane when in situ solubility isexceeded. Model-derived sulfate and methane concentration distributions combined

Fig. 3. Methane and Dissolved sulfate concentrations versus depth in the upper 1.5 m of the sedimentcolumn in 1994.

Christopher S. Martens and others—Stable isotope tracing598

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of anaerobic methane oxidation in the gassy sediments 599

with kinetic rate expressions are used to predict sulfate and methane concentrationversus depth profiles as well as the rates of the biogeochemical processes controllingthese concentrations including sulfate reduction, methane production, and methaneoxidation. Input required to run the model includes the organic carbon flux (FG) to thesediments, sediment depth, temperature, and salinity. No direct information concerningthe organic carbon flux (FG) to Eckernforde Bay sediments was available; however,measurements of sulfate pore water chemical gradients allowed Albert, Martens, andAlperin (1998) to estimate an FG value of 2.3 mol m�2 yr�1, using a simple Fick’s first lawcalculation and reaction stoichiometry assumptions as described by Martens and Klump(1984). Model results for the NRL site are illustrated in figure 5. The minimal effects ofupward advection at the NRL site in Eckernforde Bay were ignored for the purposes ofthe calculations.

The shape of the methane concentration versus depth distribution (fig. 3; seeReeburgh, 1976; Reeburgh and Heggie, 1977; and Martens and Berner, 1977) and themagnitude of the model-calculated upward methane flux indicated an important role forAMO whose predicted rates are shown in panel B of figure 5. The actual radio-tracer determined rates with a maximum rate of 16 mM yr�1 shown in figure 4 agreeremarkably well with the modeled AMO rate profile and maximum rate approaching14 mM yr�1. A first-order rate constant for anaerobic methane oxidation (kM) of 8 yr�1

was determined from model fits to the radiotracer measurements. The concentrationdistributions of dissolved sulfate and methane (fig. 5A) are well-fit by the model.However, actual in situ gas bubble distributions and concentration (Anderson andAbegg, 1997) are much more erratic as a result of unquantified bubble migration andtrapping processes.

TABLE 4

Stable carbon isotopes of CO2 in sediment porewaters at the NRL site, Eckernforde Bay

Christopher S. Martens and others—Stable isotope tracing600

METHANE STABLE ISOTOPE DISTRIBUTIONS: CONTROL BY AMO VERSUS METHANE PRODUCTION

The depth distributions of methane �13C and �D values at the NRL site areillustrated in figure 6. Two features are prominently visible in the figure 6 distributions.The first feature is the co-variation in both �13C and �D toward heavier values in theupper 20 cm of the sulfate reduction zone immediately above the zone of maximumAMO (fig. 4). This co-variation would be expected to result from fractionation occurringduring AMO (Coleman, Risatti, and Schoell, 1981). It cannot be explained as a result ofchanges in the mechanism of methane production. Changes in �13C and �D valuesassociated with variation in the proportions of acetate fermentation versus CO2 reduc-tion would be expected to produce isotopic values heavier in one of the two isotopes andlighter in the other (Whiticar, Faber, and Schoell, 1986; Burke, Martens, and Sackett,1988).

The second feature visible in the figure 6 distributions is the increase in methane�13C values below the upper 20 cm. This trend is explained by a concurrent increase in�CO2 �13C values versus depth (figure 7). The methane �13C values exhibit a relativelyconstant offset from the corresponding �CO2 values resulting from methane productionfrom an increasingly heavier �CO2 pool.

FRACTIONATION FACTORS ASSOCIATED WITH AMO IN ECKERNFORDE BAY SEDIMENTS

The dual stable isotope data collected from Eckernforde Bay during July, 1994provide an opportunity to quantify fractionation factors associated with AMO for both Cand H isotopes. In particular there is sufficient �13C and �D data for dissolved methane in

Fig. 4. Dissolved sulfate plotted versus the measured rate of anaerobic methane oxidation during 1993.

of anaerobic methane oxidation in the gassy sediments 601

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Christopher S. Martens and others—Stable isotope tracing602

the sulfate reduction zone to allow for utilizing an open system, steady-state modeldeveloped by Alperin, Reeburgh, and Whiticar (1988) for application to Skan Bay,Alaska, sediments. A kinetic isotope fractionation occurs during AMO because methanecomposed of the lighter isotopes of C and H is oxidized slightly faster than methanecomposed of the heavier isotopes (Bigeleisen and Wolfsberg, 1958). The magnitude ofthe effect is expressed as a fractionation factor, �, which is defined as the ratio of relativereaction rates of molecules containing different isotopes (Rees, 1973):

� �R/c

R*/c*(2)

where R is the reaction rate, c is the concentration of the reacting species, and the asteriskrepresents the molecule containing the heavier isotope. Fractionation factors derivedfrom the open-system model of Alperin, Reeburgh, and Whiticar (1988), should be moreaccurate for sediments from which actual isotope values have been obtained. Further-more, as they point out, there are no enrichment cultures for the anaerobic bacteriaresponsible for AMO, and, thus, no laboratory-determined values are available.

Berner’s (1980) general diagenetic equation describing the concentration-depthdistribution of pore water constituents as a function of diffusion, sediment accumulation,

Fig. 6. The �13C and �D values of methane plotted versus depth in the upper 2 m of the sediment columnin 1993.

of anaerobic methane oxidation in the gassy sediments 603

compaction, and reaction has been derived in the form of eq (3) by Murray, Grundma-nis, and Smethie (1978):

2Do

d2c

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d

dx�

oo

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dx� Rx � 0 (3)

where c is pore water methane concentration, x is depth below the sediment interface, Dois the free solution diffusion coefficient, is porosity, is burial velocity relative to theinterface, � is the depth at which the porosity gradient approaches zero, and Rx is thedepth dependent reaction rate. Alperin, Martens, and Albert (1988) have utilized eq (3)and equations derived from it in order to obtain � values associated with the process ofAMO in the anoxic sediments of Skan Bay. The equations discussed below as well asassumptions implicit in applying them are discussed in detail in their paper. Eq (3) canprovide a numerical representation of the concentration versus depth distribution ofisotopically light methane. Eq (3) can be re-written for isotopically heavy methane. Withthe additional substitution of (2) into (3) and the assumption that � is constant with depth,the equation for isotopically heavy methane becomes:

2Do

f

d2c*

dx2� 13

Dod

fdx�

oo

2 dc*

dx� 1Rx

�c2 c* � 0 (4)

Fig. 7. The �13C values of �CO2 and methane plotted versus depth in the upper 2 m of the sedimentcolumn in 1993.

Christopher S. Martens and others—Stable isotope tracing604

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of anaerobic methane oxidation in the gassy sediments 605

where f is the molecular diffusivity ratio (light:heavy) for isotopic species, and � is theisotope fractionation factor.

The first step in obtaining fractionation factors from the diagenetic equation is toinvert eq (3) and solve for the 12C reaction rate (R) needed in eq (2) above. In practice,this is achieved by differentiating a cubic spline fit to the methane concentration data inthe depth zone over which AMO occurs (the sulfate reduction zone in fig. 3) in order toobtain first and second derivatives of the methane concentration versus depth profile.These derivatives are then used to solve for the depth-dependent AMO rate, Rx:

Rx � �2Do

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2 dc

dx(5)

Model parameters utilized in eq (5) are discussed in the study site section above orappear in table 1 of Martens, Albert, and Alperin (1998) and will not be repeated here.

The cubic spline fit of the methane concentration versus depth profile obtainedduring our 1994 expedition is illustrated in figure 8A. Model-derived AMO rates versusdepth appear in figure 8B. Positive rates indicate net methane oxidation; negative ratesindicate net methane production.

Eqs (3) and (4) for isotopically light and heavy methane, respectively, were solvednumerically using a finite difference method as described by Alperin, Reeburgh, andWhiticar (1988). Solutions using various values of � were used to calculate depthdistributions of isotopically light and heavy methane. These distributions were then usedto calculate methane �13C and �D profiles as illustrated in figures 9 and 10 respectively.

Fig. 9. Sensitivity of model-predicted �13C values of methane to the magnitude of �. The different curvesrepresent predicted depth profiles for three � values over the zone of modeled net anaerobic methaneoxidation.

Christopher S. Martens and others—Stable isotope tracing606

The sensitivity of the stable isotope model to � is illustrated through comparisonwith the methane stable isotope data throughout the methane oxidation zone. The best fit� values are 1.011 � 0.001 for carbon (fig. 9) and 1.120 � 0.020 for hydrogen (fig. 10).Fractionation factors obtained for Skan Bay, the only previous site to which the modelhas been applied, were 1.0088 � 0.0013 and 1.157 � 0.023 for C and H isotopes,respectively (Alperin, Reeburgh, and Whiticar, 1988). Differences in the � values fromthe two sites are within the uncertainties estimated from the envelop of profile fits to theisotope data. This similar result is particularly interesting since the maximum rates ofAMO measured in Skan Bay sediments, 3 to 4 mM yr�1 (Reeburgh, 1980; Alperin andReeburgh, 1985), are approximately four to five times lower than the maximum ratemeasured in Eckernforde Bay. The temperature of Skan Bay sediments at the time of therate experiments was approx 4°C versus 8°C for Eckernforde Bay measurements.

CONCLUSIONS

Since the publication of alternative hypothesis (3) by Martens and Berner (1974)‘‘that methane is produced only in the absence of dissolved sulfate; however, on diffusingupward, is then consumed by sulfate reducers,’’ much progress has been made inunderstanding the microbial and biogeochemical processes controlling AMO (seereviews by Reeburgh and Alperin, 1988; Hoehler and Alperin, 1996). Furthermore, theinfluence of AMO on the stable isotopic composition of methane and �CO2 in the porewaters of marine sediments (Alperin, Reeburgh, and Whiticar, 1988) and the importantrole of AMO in the global methane budget has been recognized (Reeburgh, Whalen,and Alperin, 1993). Current kinetic models for sedimentary biogeochemical processes

Fig. 10. Sensitivity of model-predicted �D values of methane to the magnitude of �. The different curvesrepresent predicted depth profiles for three � values over the zone of modeled net anaerobic methaneoxidation.

of anaerobic methane oxidation in the gassy sediments 607

derived from the originals described by Berner (1980) are capable of accuratelypredicting the concentration versus depth distributions of methane and sulfate togetherwith the rate distributions of sulfate reduction, methane production, and anaerobicmethane oxidation (Martens, Albert, and Alperin, 1998). The use of these modelsappears required to predict the isotopic fractionation associated with AMO and thus theisotopic composition of methane and �CO2 in organic-rich sediments because of theconsortium of organisms and complexity of the biogeochemical processes involved. Weare all indebted to Bob Berner for his creative and pioneering research leadership.

ACKNOWLEDGMENTS

This study was supported by the Naval Research Laboratory as part of the CoastalBenthic Boundary Layer Project (ONR N00014-93-1-6005) and by the National ScienceFoundation Chemical Oceanography Program (OCE-9633456). Logistical support forstudies of Eckernforde Bay was provided by the Forshungsanstalt der Bundeswehr furWasserschall und Geophysik (FWG), Kiel, Germany, and the German Navy, includingdiver operations at the NRL site. We particularly wish to thank Friedrich Abegg of theUniversity of Kiel and the FWG for fruitful collaborations, Aubrey Anderson of TexasA & M University for discussions and advice during both field studies, Ingo Stender, andThomas Wever of the FWG and all the members of the CBBL group for their advice andsupport during field operations. Additional field assistance was provided by UNCgraduate students Brooks Avery and Tamara Pease during the 1993 expedition. Wethank Howard Mendlovitz for direction of the stable isotope analyses.

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