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Factors Influencing Stable Isotope Ratios in CH4 andCO2 Within Subenvironments of Freshwater Wetlands:Implications for δ-Signatures of EmissionsE. R. C. Hornibrook ab; F. J. Longstaffe a; W. S. Fyfe aa Department of Earth Sciences, The University of Western Ontario, London,Ontario, Canadab Department of Earth Sciences, University of Bristol, Bristol, UK

Online Publication Date: 01 September 2000To cite this Article: Hornibrook, E. R. C., Longstaffe, F. J. and Fyfe, W. S. (2000)'Factors Influencing Stable Isotope Ratios in CH4 and CO2 Within Subenvironments

of Freshwater Wetlands: Implications for δ-Signatures of Emissions', Isotopes in Environmental and Health Studies, 36:2,151 - 176To link to this article: DOI: 10.1080/10256010008032940URL: http://dx.doi.org/10.1080/10256010008032940

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Isotopes Environ. Healfh Stud., ZOOO, Vol. 36. pp. 151 - 176 Reprints available directly from the publisher Photocopying permitted by license only

0 2000 OPA (Overseas Publishers Association) N.V. Published by license under

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FACTORS INFLUENCING STABLE

WITHIN SUBENVIRONMENTS OF FRESHWATER WETLANDS:

OF EMISSIONS

ISOTOPE RATIOS IN C& AND CO2

IMPLICATIONS FOR S-SIGNATURES

E. R. C. HORNIBROOK*3t, F. J. LONGSTAFFEt and W. S. FYFE

Department of Earth Sciences, The University of Western Ontario, London, Ontario, Canada NdA-SB7

(Received 16 December 1999; In j k a l form 2 May 2000)

Much uncertainty still exists regarding spatial and temporal variability of stable isotope ratios (13C/’2C and D/H) in different CH4-emission sources. Such variability is especially prevalent in freshwater wetlands where a range of processes can influence stable isotope compositions, resulting in variations of up to - 50% for 6”C-C& and - 150% for 6D- CH4 values. Within a temperate-zone bog and marsh situated in southwestern Ontario, Canada, gas bubbles in pond sediments exhibit only minor seasonal and spatial variation in 6I3C-CH4, 6 D - C h and 6’3C-C02 values. In pond sediments, C02 appears to be the main source of carbon during methanogenesis either directly via COz reduction or indirectly through dissimilation of autotrophic acetate. In contrast, CH4 production occurs primarily via acetate fermentation at shallow depths in peat soils adjacent to ponds at each wetland. At greater depths within soils, CCO2 and H20 increasingly exert an influence on 6I3C- and 6D-CH4 values. Secondary alteration processes (e.g., methanotrophy or diffusive transport) are unlikely to be responsible for depth-related changes in stable isotope values of CH4. Recent models that attempt to predict 6D-CH4 values in freshwater environments from D/H ratios in local precipitation do not adequately account for such changes with depth. Subenvironments ( ie . , soil-forming and open water areas) in wetlands should be considered separately with respect to stable isotope signatures in CH4 emission models.

*Corresponding author. ‘Present address: Department of Earth Sciences, Wills Memorial Building, University

of Bristol, Queens Road, Bristol, UK BS8 1 RJ. Tel.: + 44 1 17 954 5404, Fax: + 44 I 17 925 3385, e-mail: Ed.Hornibrook@bristol.ac.uk

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152 E. R. C. HORNIBROOK er al.

Keywords: Bog; Carbon dioxide; Carbon 13; Deuterium; Ebullition; Greenhouse gases; Marsh; Methane: Methanogenesis; Sediment free-gas

1. INTRODUCTION

Atmospheric methane is a radiatively important trace gas that may be capable of altering the Earth's energy budget and contributing to global climate change [l]. Analysis of air bubbles trapped within ice cores indicates that the abundance of CH4 in the troposphere has more than doubled during the past three centuries [2-41. Identifica- tion of globally significant CH4 sources and determination of their relative strengths are critical for the evaluation of different strategies to mitigate emissions [5].

Stable isotope ratios (13C/12C and D/H) provide important constraints for the development of global CH4-budgets. Isotope mass- balance models rely upon the premise that the weighted sum of '3CH4/12CH4 and C'H3D/C'H4 ratios from all sources should be equal to that of atmospheric CH4 when corrections are made for kmetic isotope effects (KIEs) associated with CH4-sink reactions [6]. Success of the method depends upon an accurate knowledge of relative source strengths and the stable isotope composition of their emissions. The latter is expressed by convention in the standard S-notation in parts per thousand ( O k ) relative to VPDB [7]. Model estimates using this approach continue to be refined as more data become available on various CH4 sources and sinks [8 - 151. However, a wide range of 613C- and 6D-CH4 values often occurs within individual anaerobic environ- ments, which makes it difficult to estimate the average &signature of each source. Understanding the controls that govern this variability is key to refining the use of stable isotope ratios as a mass balance tool.

Natural wetlands are considered to be the largest natural contributor of CH4 to the atmosphere each year [16]. High water- table levels and an abundance of organic matter provide ideal con- ditions for anaerobic microorganisms to produce copious quantities of CH4 and C02. Wetland CH4 exhibits a wide range of variation with respect to carbon (S13C - -85 to -40%) and hydrogen (6D- -400 to - 250%) isotope compositions. Generally, this variability is attributed to KIEs associated with microbial methanogenic pathways,

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H AND C ISOTOPE RATIO VARIABILITY 153

methanotrophy and diffusion [ 17 - 201, both spatially [2 1,221 and temporally [20,23 - 251. Variations in S13C-CH4 values also may result from differences in the isotopic composition of carbon precursors (i.e., C3 versus C4 vegetation [26]). The stable hydrogen-isotope composi- tion of H 2 0 has an effect on 6D-CH4 values [27-301 because methanogenesis via acetate fermentation must derive at least one hydrogen atom from the surrounding H20 [31] and four hydrogen atoms are obtained from coexisting H 2 0 when CH4 is produced by the C02-reduction pathway [32]. Recently, de Graaf et al. [33] demon- strated that hydrogen-isotope exchange can occur between the methyl group of acetate and H20 during methanogenesis in laboratory cultures. The prevalence of such exchange in natural environments has yet to be established.

Methane is released from wetlands to the atmosphere via three different processes: (i) diffusion across the water-air interface, (ii) plant-mediated transport by passive diffusion and effusion, or active ventilation of submerged tissues, and (iii) ebullition of exsolved gas bubbles [34,35]. Upward diffusion of CH4 through wetland soils ty- pically involves exposure to methanotrophs in the acrotelm, which can result in a significant fraction of the CH4 being consumed before it reaches the atmosphere. The processes of diffusion and methanotro- phy both favour preferential removal of I2C- and 'H-bearing CH4 from soils. The presence of vascular flora usually enhances CH4 emissions from wetlands because such plants can provide a direct conduit for CH4 from the anoxic zone to the atmosphere [36]. In addition to increasing CH4 emissions, plants can also limit isotopic fractionation resulting from methanotrophy; however, transport through intercellular channels and across the leaf-atmosphere boundary may still impart an isotope effect as a result of diffusion or effusion [35].

Ebullition is the least predictable and most sporadic of the three emission modes. It can be a significant release mechanism because it is a rapid process that permits only minimal exposure of CH4 to methanotrophs. Ebullition tends to be the most common transport process during the warmest months and in the warmest climates. In tropical wetlands, it has been reported to account for as much as 70 to 80% of annual emissions [ 16,371. Bubbling events can be triggered, especially in shallow-water zones, by changes in wind velocity, water-

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154 E. R. C . HORNIBROOK et al.

tables levels and atmospheric pressure [38 -431. With the exception of pressurized ventilation in certain types of aquatic plants, ebullition is the only emission mode that consistently does not affect stable isotope ratios in CH4 [18,44]. Rapid release of gas bubbles by ebullition prevents 13C/”C and D/H ratios from being altered by KIEs associated with diffusion or the metabolism of methanotrophs. Thus, it is important to understand better the controls that govern 6I3C and dD-values of CH4 in sediment free-gas because isotope signatures in such CH4 are likely to be expressed in emissions to the atmosphere.

The primary objective of this study was to investigate factors that influence stable isotope ratios in CH4 and C 0 2 from a temperate-zone bog and marsh. We compare the stable isotope compositions of CH4 and C02 in sediment free-gas from wetland ponds with &values for dissolved CH4 and CC02 in porewaters within peat soils adjacent to the ponds. We show that CH4 and C02 within subenvironments in wetlands can exhibit significant, but predictable differences in their stable isotope compositions. We also demonstrate that equations recently proposed to predict GD-CH4 values within freshwater environments based upon bD-values of regional precipitation should be used with caution. The equations do not adequately consider the distribution of SD-CH4 values within wetland soils and pond sediments, and cannot be used to predict 6D values of sporadic and often significant emission events that release CH4 from deep soil layers.

2. STUDY SITES

The Sifton Bog and Point Pelee Marsh are located within the Eastern Temperate climatic zone of southwestern Ontario, Canada (Fig. 1). The zone is characterized by mild winters (mean daily January tem- perature -9’C), warm summers (mean daily July temperature 20°C) and a high annual input of precipitation (- 35 cm/yr as rainfall and N 2lOcm/yr as snowfall) [45].

The Sifton Bog (43’00’00”N) is an acidic Sphagnum peat bog located within the City of London, Ontario, Canada. The bog covers an area of -28 hectares and was formed by lakefill of a small kettle depression created at the end of the last glaciation. The site consists of

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H AND C ISOTOPE RATIO VARIABILITY 155

FIGURE 1 Map showing location of the Sifton Bog and Point Pelee Marsh in southwestern Ontario, Canada.

a small central open pond (Redmond’s Pond; pH - 5.4) surrounded by a floating Sphagnum-mat (surface water pH - 3.8) that gradually changes at its periphery into lower damp woods and eventually upland wooded slopes. The flora within each of the vegetation zones have been described in detail elsewhere [46,47]. The central portion of the bog contains as much as 10m accumulation of peat [48]. The organic soils overlie deltaic deposits of sand and gravel, which in turn overlie glacial tills of a terminal moraine.

The Point Pelee Marsh (41’58’00”N) is a freshwater wetland located within Point Pelee National Park, near Leamington, Ontario, Canada. It exists within a quasi-symmetrical cuspate foreland that projects - 15 km into Lake Erie. Sand ridges that enclose the marsh formed - 4000 years B.P. from remnants of the Pelee-Lorain moraine during a progressive rise of lake levels in the western and central basins of Lake Erie [49,50]. Marsh waters are considered to be isolated from those of Lake Erie, except during major storm events [51,52]. The present marsh covers an area of - 1,050 hectares or - 70% of the park area. It consists primarily of extensive floating-mats (surface water pH - 5.7)

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156 E. R. C. HORNIBROOK el al

bearing monocultures of the Common Cattail (hybrid Typha x glauca Godr.) with minor subpopulations of Typha ungustifolia (L.) and T. lutifolia existing in restricted areas [51]. The Typha-bearing mats are interspersed with large, shallow open ponds and channels (pH N 6.9) that contain an abundance of aquatic vegetation during summer months.

3. METHODS

Sediment free-gas was obtained from ponds by disturbing organic-rich bottom sediments with a long pole. Typically, water depth at the sites was greater than 2 meters. The gas bubbles released were trapped in an inverted, gas-tight, water-filled, plexiglass box. The top of the box was fitted with a valve through which the sediment free-gas was transferred to HgC12-treated syringes that were fitted with a three-way stopcock. Samples were stored on ice further to inhibit microbial activity during transport to the laboratory. Water for 6D-H20 analyses was obtained from bottom sediments using a sipper. Samples were collected by drawing water into syringes that were fitted with a three-way stopcock. Porewater for analyses of dissolved CH4 and CC02 was sampled from floating-mat soils in a similar manner. These latter samples were treated with HgC12 to inhibit microbial activity.

Concentrations of C02 and CH4 in sediment free-gas and headspace samples stripped from porewaters were determined using a Hewlett Packard 5890A gas chromatograph. Separation of C 0 2 and CH4 was performed at 35°C on a 4m x 3.2mm OD stainless steel column packed with Porapak@ QS coated on 80/100 mesh particles. Samples were injected onto the column using a gas-sampling valve fitted with a 0.25 cc sample loop that was maintained at a temperature of 50 f 1°C. Sample COz was reduced to CH4 on a NiO catalyst at 325°C to permit the measurement of both components using a flame-ionization de- tector (FID). The relative precision of C02 and CH4 analyses was better than f 1%.

The C02 in sediment free-gas was prepared for isotopic analysis by extraction under vacuum at liquid-nitrogen temperatures. The residual CH4 was converted to C02 and H20 on CuO at 880°C. Dissolved CH4 and CC02 were extracted from porewaters for isotopic analysis by

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H AND C ISOTOPE RATIO VARIABILITY 157

vacuum sparging of acidified samples with a UHP helium/oxygen blend (85 : 15). The C02 and CH4 were removed from the sparging effluent using the same vacuum extraction line and method described for sediment free-gas. Yields from extracted CO:! and CH4-derived C 0 2 were measured by manometer. Combustion yields of CH4 from a Research Grade CH4 standard were 99 i 1 %. Both CH4-derived and porewater H 2 0 were converted to H2 by the zinc-reduction method [53]. Stable isotope compositions are reported in the standard 6- notation: S(%O) = ((Rsample/Rstandard)- 1) x 1000 where R is D/H or l3C/l2C [7]. Isotopic results for carbon and hydrogen are reported relative to VPDB and VSMOW, respectively [54]. The precision of 613C analyses for C 0 2 and CH4 was better than i 0.2%, while SD measurements for CH4 and H 2 0 had precisions, respectively, of i 3% and f 2%0 based upon multiple analyses of standards.

4. RESULTS AND DISCUSSION

The concentrations and stable isotope compositions of CH4 and C 0 2 in gas bubbles collected from bottom sediments in ponds at the Sifton Bog and Point Pelee Marsh, and the 6D values of coexisting water are presented in Table I. Relationships observed among these data are discussed below along with a comparison to previously reported [21,22] stable isotope values for CH4 and CCOz dissolved in porewaters within soils at each wetland.

4.1. CH4 and COz Concentrations

The abundance of CH4 versus C 0 2 within sediment free-gas from each wetland is shown in Figure 2. The average concentration of CH4 within gas bubbles from the two wetlands is approximately the same (Sifton Bog [CH4] = 69.9 i 11.9 mole%; Point Pelee Marsh [CH4]=69.2 3= 12.8mole%). The high concentration of CH4 in se- diment free-gas results from high rates of primary productivity in ponds (i.e., an abundance of plant debris) coupled with an absence of O2 or alternative electron acceptors (e.g., SO:-, NO;, etc.) in organic- rich bottom sediments. The low solubility of CH4 in water promotes efficient transfer from porewaters to the gaseous state.

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158 E. R. C. HORNIBROOK ef al.

TABLE I Concentrations of CH4 and C 0 2 and stable isotope compositions of CH4, C02 and co-existing H20 in bottom sediments of ponds at the Sifton Bog and Point Pelee Marsh, southwestern Ontario, Canada

Sampling CH, C02 d3C-CH4 6D-CH4 6’3C-C02 6D-H20 date (mol YO) (mol YO) (%) (x) (%I

~~~~ ~ ~ ~ ~~ ~ ~

Sifton Bog Nov. 94 66.2 2.9 - 58.6 - 341 - 5.2 - 65

62.6 0.8 - 63.4 - 309 + 0.5 -

Feb. 95 62.6 0.8 - 63.4 - 309 -0.5 - 62 Jun. 95 76.0 6.4 - 62.7 - 282 + 0.9 - 70

- - 0.5 79.0 5.9 - 62.4 - 68.7* 6.6 - 62.0 - 245 - 0.8 -

70.7* 5.3 - 62.5 - - 0.4 -

82.0 6.6 -61.7 - 308 - 0.9 -

77.4 3.4 - 62.6 - 303 + 1.1 -

Aug. 95 83.4 7.8 - 60.8 - 291 i 2.6 - 68

Nov. 95 38.5 3.5 - 60.2 - 319 - 1.3 - 70

Mar. 96 74.0 3.5 - 62.9 - 301 - 2.7 - 70 Aug. 96 58.6 5.4 - 62.1 - 335 -4.5 - 67

78.6 5.7 -63.1 - 297 0.0 -

Point Pelee Marsh - - 5.2 Sept. 94 75.6 4.0 - 62.1 -

71.9 3.4 - 65.5 - - 5.2 -

Nov. 94 29.7 0.7 -61.4 - 305 - - 47 67.4* 0.9 - 66.3 - 276 - 2.0 - 65.8* 0.6 - 66.7 - 267 - 1.4

66.3* 0.8 - 70.4 - 281 - 5.4 -

61.9 0.7 - 68.8 - 290 - 8.4

83.2’ 3.8 - 62.6 - 293 -5.1 -

85.3 2.5 - 62.7 - 303 - 4.6 -

-

Apr. 95 63.5* 1.1 - 69.5 - 289 - 5.2 - 56

-

Sept. 95 83.2* 3.3 -61.6 - 291 - 5.3 - 42

Nov. 95 69.0 1.2 - 64.8 - 275 - 1.8 - 50 73.5* 3.1 - 65.6 - 279 - 0.7 - 72.3* 2.1 - 65.1 - 275 -0.5 -

63.8 5.0 - 63.7 - 307 -4.5 -

Mar. 96 75.3 5.4 - 65.2 -281 - 1.4 - 54

* Replicate analyses.

The average concentration of C 0 2 is greater within gas bubbles from the Sifton Bog ([CO,] = 4.6 f 2.2 mole%) than the Point Pelee Marsh ([CO,] = 2.4 f 1.6 mole%). The higher abundance of COz in sediment free-gas from the bog environment can be readily explained by differences in porewater pH. Water typically has a pH of -5.2 within bottom sediments at the Sifton Bog. In contrast, porewater in bottom sediments at the Point Pelee Marsh has a pH of -6.7.

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H AND C ISOTOPE RATIO VARIABILITY 159

n

$ 5 W e

E 4 0 2

1

0- 0 25 50 75 100

CH, (mole %)

FIGURE 2 Concentrations of CO2 versus CH4 within gas bubbles collected from sediments in Redmond’s Pond at the Sifton Bog (0 ) and channels at the Point Pelee Marsh (A). The smaller, solid symbols with an enclosing box represent the average and standard deviation (lu) of concentration measurements for C02 and CH4 at each wetland.

Equilibrium partitioning of C02(aq) and HCO; is very sensitive within the pH range of 5 to 7. A lower pH results in a greater fraction of dissolved C 0 2 existing as C02(,,, and consequently, as C02(,, [55].

Despite the lower concentration of C02 in sediment free-gas, dissolved C C 0 2 is generally more abundant within the marsh pore- waters. Under the same conditions of pressure (water depth= 2m) and temperature (15”C), the average abundance of C02 within gas bubbles from each wetland corresponds to a C C 0 2 concentration of -2700vM for the Sifton Bog and -3900pM for the Point Pelee Marsh. These calculated values are consistent with measurements of CC02 concentrations in porewaters reported previously for each wetland [21]. Higher rates of decay likely contribute to the greater overall abundance of C 0 2 in the marsh environment.

4.2. 613C Values for CH4 and G)COz

Stable carbon-isotope values for C 0 2 and CH4 extracted from sediment free-gas are shown in Figure 3a. Using a-notation, the

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160 E. R. C. HORNIBROOK ef al.

10

- 0 3

?

v r g -10

L. -20

-30 -90 -80 -70 -60 -50 -40 -30

&')C-CH, (%o)

FIGURE 3 6I3C values of (a) C02 and C& in sediment free-gas from the Sifton Bog (0) and Point Pelee Marsh (A), and (b) CC02 and CH, in porewaters collected from Sphagnumsoils at the Sifton Bog @) and Typha-soils at the Point Pelee Marsh (+ ). The smaller, solid symbols with an enclosing box in (a) represent the average and standard deviation (lo) of measured 6°C values in sediment free-gas at each wetland. Stable carbon-isotope values in (b) were reported previously by Hornibrook ef al. 121,221. Solid lines passing through the porewater data were determined by linear regression. The arrows indicate direction of increasing sample depth in peat soils. Values of depth were measured with respect to the mat surface. Lines of constant ac delineate fields where 6I3C-CH4 and d3C-C02 pairs have been suggested by Whiticar et al. [17] to represent CH4 production by acetate fermentation (- 1.040 to 1.055) and C02-reduction (- 1.055 to 1.090).

magnitude of stable carbon-isotope fractionation between COz and CH4 is defined by:

(S13CCS + 1000) (6'3CCH, + 1000)

ac =

Normally, this formulation of a is used to express isotope fractiona- tion associated with equilibrium processes. Since isotope effects

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H AND C ISOTOPE RATIO VARIABILITY 161

associated with biological production of CH4 are primarily kinetic in origin, our use of Eq. (1) to calculate ‘apparent’ a-values is merely a convenient way to describe isotope separation between C 0 2 and CH4. Lines of constant a, shown in Figures 3a and b delineate fields where 6I3C-CO2 and 6I3C-CH4 values have been suggested by Whiticar et af. [I71 to represent CH4 production by acetate fermentation (w 1.040 to 1.055) and COz reduction (- 1.055 to 1.090).

Values of 6I3C for C 0 2 and CH4 in sediment free-gas plot entirely within the C02-reduction field (Fig. 3a); however, it is also possible that the CH4 was formed via acetate fermentation. Autotrophic (ie. , anabolic) acetogens that produce acetate from C 0 2 using H2 or formate as a reductant have a significant KIE of -60%0 associated with their metabolism [56]. Since C 0 2 is abundant within wetland porewaters, the full magnitude of the KJE is expressed, yielding acetate with a very low ~5’~C-value. The amount of acetate contributed by autotrophic acetogens to porewaters in freshwater systems is poorly known [57]. Wood and Ljungdahl [58] estimate that - 10% of acetate produced in anaerobic environments may be formed from porewater CO2 via autotrophic acetogenesis. Refinement of this figure awaits further study. Regardless, 6I3C values for C02 and CH4 shown in Figure 3a suggest that a significant fraction of carbon is being derived from COz during methanogenesis within sediments in wetland ponds. However, it is unclear whether the carbon from C02 is being in- corporated directly into CH4 via methanogenesis by the C02-reduction pathway or indirectly through fermentation of autotrophically-formed acetate.

The lines labelled ‘Sifton Bog’ and ‘Point Pelee Marsh’ in Figure 3a were determined by linear regression of 613C values for CC02 and CH4 from porewaters within soils adjacent to ponds at each wet- land (data shown in Fig. 3b). The linear trend of the 6I3C pairs with depth is interpreted to result from a shift in the dominant pathway for methanogenesis [21,22]. In shallow soils where labile organic substrates are most abundant, CH4 production should occur mainly by fermentation of acetate that has been produced from the decay of organic matter. Such an interpretation is supported by the presence of CH4 that exhibits I3C-enrichment and low 6D-CH4 values (< -340%0); the latter indicates that the CH4 has not been subjected to significant methanotrophy. Hence, the high 6l3C-CH4 values must

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162 E. R. C. HORNIBROOK et al

reflect either the small KIE associated with acetate fermentation [59] or minimal expression of this KIE because of low concentrations of acetate in porewaters [60]. Intersection of the regression curves at 613C-CC02 = - 21.3Y~ and 613C-CH4 = -42.3%0 further supports the assertion that acetate is an important substrate for methanogenesis at shallow depths [22]. These values of 6I3C are similar to the intramolecular distribution of carbon isotopes within acetate that has been produced by anaerobic decay of C3 vegetation: 6I3C- COO- = -24.4 to -22.9% and 6I3C-CH3- = -42.9 to -39.7%0 (Fig. 3b, star symbols) [61]. A low concentration of acetate in porewaters will result in minimal expression of the KIE associated with acetate dissimilation and consequently, production of CH4 and C02 with 6I3C values that reflect the internal distribution of carbon isotopes in acetate. Acetate fermentation in anaerobic incubations of fresh peat has been reported to yield CH4 with 613C values of -44.3 f 15.2'360 and -44.5 f 6.4Oh [20]. Regressions curves deter- mined from 613C-values for CC02 and CH4 from porewaters in two Minnesota peatlands [62] also intersect at approximately the same 613C-values for CH4 and CCOz [23].

Methanogenesis within deeper soils gradually favours the CO2- reduction pathway or possibly, the dissimilation of autotrophic acetate. Such a change is reflected by the increase of cyc values with depth, resulting from progressive 13C-depletion in CH4 and I3C- enrichment in CC02 (Fig. 3b). Similar trends in 6I3C values for CH4 and C02 can occur temporally in shallow soils of peatlands. Avery et al. [20] used I4C-labelled substrates to confirm that seasonal changes in 6I3C-CH4 values at shallow depths in a peat bog were strongly affected by the prevalence of methanogenesis via C02 reduction and acetate fermentation. A small fraction of the change in aC values with depth may also result from lower temperatures in deeper soils, which can cause an increase in the KIE associated with the C02-reduction pathway [63,64]. However, metabolic control theory predicts that increased recalcitrance of an organic substrate through microbial decay will shift CH4 production to involve greater utilization of H2 [65]. Briefly, labile substrates are most abundant in shallow soils and fermentation of biopolymers proceeds readily, especially during the warmest months. Under such conditions, methanogenesis is the rate- limiting step in the anaerobic chain of decay. Efficient decay of

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H AND C ISOTOPE RATIO VARIABILITY 163

monomers produced by the fermentation of complex substrates requires that the concentration of H2 in porewaters be maintained at very low levels in order for the metabolism of H2-producing syntrophs to remain exergonic [66]. The short-term inability of H2-oxidizing methanogens to sustain the appropriate levels of H2 results in greater production of acetate. Consequently, when labile substrates are abundant, methanogenesis is favoured by the acetate fermentation pathway. As decay continues and organic matter becomes more recalcitrant, fermentation of complex biopolymers gradually becomes the rate-limiting step in the anaerobic chain of decay. Under such conditions, H2-oxidizing methanogens are capable of maintaining low levels of H2. Although the steady-state concentration of H2 then decreases in porewaters, a greater amount of H2 is being cycled through the system. The result is enhanced production of CH4 via H2 oxidation coupled to C 0 2 reduction at depths where soil organic matter is more refractory [65].

In contrast to peat soils, carbon-isotope separation is relatively constant between C02 and CH4 in gas bubbles from bottom sediments in ponds: Sifton Bog ac= 1.0652 f 0.0032 and Point Pelee Marsh ac = 1.0659 * 0.0037. These ac values suggest that the proportion of methanogenesis involving porewater C02 as a carbon source may be similar in pond sediments at each wetland. However, it is important to consider the 6I3C value of total C 0 2 in porewaters rather than just that of C 0 2 within gas bubbles because the latter will be influenced by porewater pH. A higher pH results in a greater proportion of HCO; within porewaters and consequently, a more positive 613C-value for CCO? relative to CO,,,,. The SI3C value of CH4 is unaffected because significant isotopic fractionation does not occur during dissolution of CH4. Values of 6I3C measured from CH4 in sediment free-gas and calculated values of 613C-CC02 corrected for pH effects, yield ac= 1.0647 and 1.0718, respectively, for the Sifton Bog and Point Pelee Marsh. The pH-corrected values of ac suggest that utilization of porewater C02 during methanogenesis may be greater at the Point Pelee Marsh, consistent with findings that higher rates of decomposi- tion typically occur in more minerotrophic wetlands such as marshes

Avery et al. [64] reported that CH4 within organic-rich sediments from a freshwater estuarine wetland exhibited only minor seasonal

1671.

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164 E. R. C. HORNlBROOK er al.

variations in SI3C values (- 6%). Methanogenesis occurred predomi- nantly via C 0 2 reduction in the sediments and significant seasonal shifts could not be detected in the prevalance of methanogenic pathway. Using I4C-labelled substrates, it was demonstrated that over half of the increase in S13C-CH4 values during summer months could be attributed to increases in S'3C-CC02 values resulting from higher rates of I2CO2 consumption and a decrease in the magnitude of the KIE associated with the C02-reduction pathway [64]. We note several interesting similarities between methanogenesis in the estuarine wetland and pond sediments from the wetlands described in this study. First, at the Sifton Bog and Point Pelee Marsh, values of 613C for sediment free-gas CH4 and C02 also exhibit only minor seasonal and spatial variations, and suggest a predominance of methanogenesis via the C 0 2 reduction pathway. Second, the estuarine sediment was described as an organic-rich 'muck', which is texturally and com- positionally very similar to material at the bottom of ponds at the Sifton Bog and Point Pelee Marsh. Notably, the main contributors of organic matter to sediments are the same in each of these wetlands: submerged aquatic macrophytes and algae.

In contrast to the situation for the sediment free-gas from the ponds, much greater seasonal and spatial variability has been reported for SI3C-CH4 and 6I3C-CCO2 values in soils-forming areas of peatlands [20-221. It is possible that distinctly different processes control 613C values within pond sediments versus peat soils in the same environ- ment. Within peat soils, large changes in 6I3C-CH4 and 613C-CC02 values appear to result from differences in the prevalence of methanogenic pathway [20-221, while the smaller changes in 613C values within pond sediments may be governed by the effects of temperature variations on rates of methanogenesis (i.e., consumption of '2C02) and the magnitude of the KIE associated with the C02- reduction pathway [MI.

Differences in S13C values for CH4 and C02 from ponds versus soils highlight the importance of considering subenvironments separately when modelling trace gas emissions from wetlands. Methane released from ponds by ebullition is likely to have a 6I3C value that is significantly different from that emitted from soil-forming areas. Ebullition is a rapid process that prevents methanotrophs from altering primary 6I3C-signatures of CH4 in sediment free-gas [18]. In

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H AND C ISOTOPE RATIO VARIABILITY 165

contrast, CH4 released from soils typically must pass through an aerobic surface-layer with a thickness that will vary depending upon the depth of the water table. At the Sifton Bog and Point Pelee Marsh, CH4 in soils is I3C-enriched relative to that within gas bubbles in pond sediments because of a greater prevalence of the acetate fermentation pathway. Methanotrophy during transport to the atmosphere may further serve to increase 613C-CH4 values. Methane can be released from wetland soils by ebullition or plant-mediated transport without isotopic fractionation; however, the 6I3C value of such emissions will still be higher than that from adjacent ponds assuming that CH4 and C02 in soils exhibit a S'3C-distribution similar to that shown in Figure 3b.

4.3. 6D Values for CH4 and H 2 0

Solid lines numbered 1 through 6 in Figures 4a and b represent equations that have been suggested to describe relationships between SD-CH4 and 6D-H20 values in nature and laboratory incubations [17,29,30,68]. A slope of 0.250 (line 4) represents the minimum possible hydrogen isotope interdependence between CH4 and H 2 0 , corresponding with 1/4 of hydrogen atoms within CH4 being derived from coexisting water. Such conditions have been suggested to be associated with methanogenesis via acetate fermentation when three hydrogen atoms are transferred intact from the methyl group of acetate to CH4 [17]. Recently, it was demonstrated that H-isotope exchange can occur between the methyl group of acetate and ambient H 2 0 [33]. However, the magnitude and rate of exchange under in situ conditions remain largely unknown. Hydrogen-isotope exchange between the methyl group of acetate and coexisting H20 will shift SD-CH4 to higher values because ambient water typically is D- enriched relative to microbial CH4. Line 1, which has a slope of unity, is considered to represent the upper limit for interdependence between SD-CH4 and 6D-H20 values in natural environments 1171. Methane formed via C 0 2 reduction must acquire all four hydrogens from ambient H20 [32] with an associated hydrogen-isotope fractionation of - - 180 f lo!% (Le., the intercept of line 1) [17]. However, acetate produced by autotrophic acetogens also may exhibit deuterium- enrichment within the methyl group [69]. Subsequent dissimilation of

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166 E. R. C. HORNBROOK et al.

-200 (a) sediment free-gas

f -400 I I I I , 1 1 , , , , , , , , , , , , , I I u

-200 KQ

-250

-300

-350

-4no -100 -80 -60 -40 -20 0

6D-H,O (%o)

FIGURE 4 6D-CH4 versus 6D-H20 values for (a) gas bubbles and coexisting water from sediments in Redmonds Pond at the Sifton Bog ( 0 ) and channels at the Point Pelee Marsh (A), and (b) dissolved CH4 and porewater from Sphagnum-soils at the Sifton Bog (X) and Typha-soils at the Point Pelee Marsh (+). The smaller, solid symbols with an enclosing box in (a) represent the average and standard deviation (lo) of measured 6D- values. Stable isotope data in (b) were reported previously [21]. The equations that correspond with numbered lines in the figure have been suggested to describe natural relationships between 6D-CH, and coexisting 6D-H20 values [17,29,30,68]. The dotted lines on either side of line 5 represent the 95% confidence interval for the data that define this relationship (291.

1. 6D-CH4 = 1 .OOO 6D-H20 - 180( f lo)% + C02 reduction - in siru [ 1 1 2. 6D-CH4 = 0.675( i 0.10) 6D-Hz0 - 284( f 6)'A -P Global freshwater - in siru 1301 3. 6D-CH4 = 0.437( 0.05)6D-H20-302( i 15)% -+Acetate fermentation - in vivo

4. bD-CH, = 0.2506D-Hz0- 321°% -+Acetate fermentation - in siru [17] 5 . 6D-CH4 = 0.444( 0.03) 6D-Hz0 - 321 ( f 4)% + Mesophilic incubations- in vivo

6. 6D-CH4 = 0.683( f 0.02)6D-H20- 31 7( f 20)%- COz reduction - in vivo [68]

[681

~ 9 1

autotrophically formed acetate by methanogens could contribute to higher GD-CH4 values. However again, the relative contribution of acetate to wetland porewaters by autotrophic acetogens is poorly known [57].

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H AND C ISOTOPE RATIO VARIABILITY 167

Line 6 is based on results from laboratory cultures that were interpreted to have undergone a shift in methanogenesis from acetate fermentation to the C02-reduction pathway, as deduced from changes in 6I3C-CH4 values 1681. At present, it is questionable whether GD-CH4 values from laboratory incubations dominated by C02 reduction can be extrapolated to natural environments. Elevated H2-levels within short-term cultures may influence 6D-CH4 values because the enzyme hydrogenase is capable of mediating hydrogen exchange between intracellular H20 and H2 [70]. The rate of hydrogen exchange mediated by hydrogenase is directly proportional to H2 abundance [71]. Because microbial Hz tends to be extremely depleted of D, CH4 may be produced with abnormally low GD-values [70]. In natural environments, intracellular hydrogen exchange between H2 and H 2 0 typically is unimportant because the H2 concentration is very low. However, the concentration of H2 within laboratory cultures typically is 3 to 5 orders of magnitude greater than within natural environments such as wetland soils.

Lines 3 and 5 also were determined using GD-CH4 and GD-H20 values obtained from anaerobic incubations [29,68]. The culture media in each case contained an abundance of labile substrates, re- sulting in a predominance of the acetate-fermentation pathway. Cultures used for line 5 were amended with butyrate and were also incubated in the mesophilic range, which strongly favours the pro- duction of acetate by catabolic anaerobic microorganisms. The simi- larity between lines 3 and 5 on one hand, and line 4 on the other hand, is not unexpected since methanogenesis via acetate dissimilation should predominate in the mesophilic cultures. One noteworthy point is that although the incubations were dominated by acetate fermenta- tion and utilized a range of 6D-H20 compositions, substantial (i.e., measureable) hydrogen exchange between the methyl group of acetate and ambient water was not apparent from the GD-CH4 values. The GD-CH4 and 6D-H20 values from the incubations followed approxi- mately the same relationship (line 4) reported by Whiticar et al. [I 71 for methanogenesis via acetate fermentation. Moreover, methanogenesis occurred in the mesophilic incubations at temperatures that are well above the normal range that exists in natural freshwater environments. The higher temperatures should have enhance rates of hydrogen- isotope exchange between the methyl-group of acetate and H 2 0 if the

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168 E. R. C . HORNIBROOK ef al.

process is a significant one during methanogenesis via acetate dissimilation.

4.3.1. Sediment Free-gas

The D/H ratio in ambient H20 clearly influences SD-CH4 values within natural systems. Such a relationship is evident in Figure 4a, which shows 6D values for CH4 and H 2 0 from sediments in ponds at the Sifton Bog and Point Pelee Marsh. The large open ponds in the marsh environment are more susceptible to evaporation, which results in greater D-enrichment of this water. Consequently, the average 6D- value of CH4 in gas bubbles at the Point Pelee Marsh (solid triangle) is higher than that from the Sifton Bog (solid circle). The positioning of 6D pairs for CH4 and H 2 0 above line 2 is unlikely to be the result of alteration effects. Microbial oxidation and diffusive transport can cause an increase in SD-CH4 values; however, these processes will simultaneously affect S13C-values. Methane in sediment free-gas does not exhibit any significant enrichment in 13C as indicated by low 6I3C- values (k., < -6OX; Fig. 3a). Moreover, the gas bubbles contain a high concentration of CH4 (- 70 mol%) and a low concentration of C 0 2 (-2 to Smol%), which is inconsistent with widespread me- thanotrophy. In addition, CH4 is released from bottom sediments primarily by ebullition, a process that is known to be non- fractionating with respect to stable isotopes [I 81.

4.3.2. Dissolved CH4

Values of SD for dissolved CH4 and coexisting H 2 0 from peat soils at the Sifton Bog and Point Pelee Marsh are shown in Figure 4b. Values of 6D-CH4 increase significantly with depth in each wetland despite only small variations in 6D-H20. The SD pairs in shallow porewaters coincide with the lines 2 through 5. At shallow depths, organic matter is most labile, promoting the prevalence of methanogenesis by acetate fermentation. This behaviour was also indicated by SI3C-CH4 and Si3C-CC02 values in shallow soils (Fig. 3b). Not surprisingly, lines 3 and 5 have been determined from incubations that were rich in labile substrates, and lines 2 and 4 are based largely upon SD values from CH4 and H 2 0 collected at shallow depths in freshwater environments.

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H AND C ISOTOPE RATIO VARIABILITY 169

The trend to higher SD-CH4 values with increasing depth (Fig. 4b) can be interpreted as a shift in the predominance of methanogenic pathway from acetate fermentation to C 0 2 reduction [17]. Such a change is supported by the trend of 613C-CH4 and 6’3C-CC02 values across the same range of depths (Fig. 3b). Once again, the potential role of autotrophic acetogens must also be considered since microbial reduction of C02 may produce acetate with a methyl group that is enriched in deuterium [69]. As noted earlier, carbon and hydrogen isotopes cannot be used, either together or separately, to uniquely distinguish CH4 that has been produced via the C02-reduction pathway or through dissimilation of autotrophic acetate. The trend of GD-CH4 values with depth suggests that hydrogen derived from H 2 0 is being incorporated increasingly into microbial CH4; however, it remains to be determined whether the mechanism for uptake is direct reduction of C 0 2 to CH4, dissimilation of autotrophic acetate, or possibly, H-isotope exchange between ambient H20 and acetate produced by catabolic decay [33].

4.3.3. Alteration: Methanotrophy and Diflusion

The equation defining line 5 that was determined from mesophilic incubations has been suggested to describe a natural relationship between 6D-CH4 and bD-H20 values in shallow freshwater environ- ments [30]. In addition, values of GD-CH4 that deviate from this rela- tionship in natural systems have been suggested to result from secondary alteration changes in the predominance of methanogenic pathways were considered unlikely to influence SD-CH4 values [30].

Methanotrophy and diffusive transport are the only alteration processes that occur within wetland environments that are known to cause a significant increase in SD-CH4 values. Let us consider whether either or both processes may have affected the results shown in Figure 4b. First, in soils at the Sifton Bog and Point Pelee Marsh, dissolved 0 2 is depleted from porewaters situated several centimeters below the surface of the water table [22]. Typically, the water table is at or just below ground level at each wetland. Dissolved sulfate (SO:-) is present in only minor quantities ( ie . , < lOOpM) in sur- face waters. The abundance of SO:- decreases to < 5 pM within porewaters situated -1 5 cm below the water table. Porewater CH4 that

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170 E. R. C . HORNIBROOK et a/ .

occurs below 15 cm depth cannot be subjected to methanotrophy in the absence of suitable electron acceptors.

Second, isotopic fractionation associated with diffusion is caused by the preferential transport of isotopic species that have a lower mass ( i e . , '*C and 'H). The concentration of CH4 in porewaters increases with depth in both wetlands, which results in upward diffusion of CH4 to the atmosphere. Slow upward transport of CH4 may ex- plain an increase in 6D-CH4 values with depth (Le., preferential movement of C'H4 versus CH3D); however, 6I3C-CH4 values de- crease across the depth interval in both wetlands (Fig. 3b). A molecular transport process that discriminates purely on a mass basis should affect 13CH4 (17.035 amu) and 12CDH3 (17.037amu) in a similar manner. The porewater distributions of 6I3C-CH4 and 6D-CH4 values in these wetland soils cannot be accounted for by either diffusion or methanotrophy.

4.3.4. Predicting GD-CHd from GD-HZO

The relationship between 6D-CH4 and 6D-H20 values described by lines 2 through 5 (Fig. 4) are adequate for many freshwater environments because methanogenesis at shallow depths typically will be dominated by acetate fermentation. Under normal hydrologic conditions, the majority of CH4 released to the atmosphere will be produced in a shallow zone - 15 cm below the surface of the water table [72]. Hence, a knowledge of SD-H20 values for a region could permit 6D values of CH4 emissions to be approximated using these relationships [30]. However, 6D values of CH4 at greater depths within wetland soils will deviate strongly from those predicted by such equations because D/H ratios in porewater increasingly exert an influence on GD-CH4 values as decay proceeds and organic substrates age. Whether the mechanism is primary ( i e . , pathway driven) or secondary (i.e., hydrogen-isotope exchange) is presently unclear. Regardless, ebullitive release of gas stored at depth in wetland soils will produce CH4 emissions with GD-values that cannot be predicted using the relationships that define lines 2 through 5. Such emission events occur as a result of a reduction in confining pressure during periods of low water-table levels [40-42,731 and during sudden

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H AND C ISOTOPE RATIO VARIABILITY 171

decreases in atmospheric pressure associated with obtruding storm- fronts [39,43]. These sporadic events can account for a significant portion of annual CH4-emissions from wetlands [16,41,42,73].

5. SUMMARY

The stable isotope compositions of CH4 and C02 are strongly influenced by conditions within different subenvironments in fresh- water wetlands. Less variation occurs in 6I3C values for CH4 and C02 from gas bubbles in pond sediments than from porewaters in soils situated adjacent to the ponds. Methanogenesis by the C02-reduction pathway or possibly, through dissimilation of autotrophic acetate, predominates in pond sediments. In contrast, CH4 production in peat soils proceeds primarily via acetate fermentation at shallow depths, shifting to a greater dependence upon porewater C02 as a carbon source in deeper soil-layers as organic matter becomes increasingly recalcitrant during substrate ageing. Consequently, 6'3C-CC02 and 613C-CH4 values in shallow soils are controlled primarily by 13C/12C ratios within organic compounds derived from the decomposition of vegetation. At greater depths, large KIEs associated with microbial utilization of C C O ~ cause an increase in 613c-cco2 values (i.e., preferential utilization of 12C02) and a decrease in 613C-CH4 values (Le., production of 12CH4). In contrast, 613C values of CH4 and C02 in pond sediments appear to be controlled primarily by the microbial KIE associated with C02-utilization. It is possible that seasonal variations in temperature also influence 6I3C-CH4 values through changes in rates of l2CO2 consumption and the magnitude of the KIE associated with the C02-reduction pathway, especially within pond sediments.

The 6D values of H20 exert varying degrees of influence upon 6D- CH4 values in wetland environments. Differences in evaporative enrichment of H20, and hence 6D-H20, are reflected in differences in 6D-CH4 values between wetlands from the same geographic area. The 6D values of CH4 in gas bubbles from pond sediments exhibit less variation than the bD values of CH4 dissolved in porewaters with- in peat soils. At shallow depths in soils, methanogenesis occurs predominately via acetate fermentation and 6D-CH4 values are

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172 E. R. C . HORNIBROOK ez al.

affected only to a minor extent by D/H ratios in H20. Within deeper soil layers, H 2 0 exerts a greater influence on SD-CH4 values; however, it is unclear whether the mechanism involves microbial processes (it., C 0 2 reduction or dissimilation of autotrophic acetate) or hydrogen- isotope exchange between H 2 0 and the methyl group of acetate. Changes with depth of SD-CH4 (and SI3C-CH4) values in soils cannot be attributed to alteration by methanotrophy or diffusive transport.

Differences in the stable isotope compositions of CH4 and C 0 2 within subenvironments in freshwater wetlands are likely to be expressed in trace gas emissions. Methane is released from ponds primarily by ebullition. Since the process is rapid and does not affect isotope ratios, CH4 emissions from ponds will retain a I3C-depleted signature. In contrast, CH4 is produced in shallow soils predominate- ly via acetate fermentation, resulting in higher ~5~~C-values. In addi- tion, CH4 released from soils typically must pass through an oxic surface-layer rich in methanotrophs, providing further opportunity for 13C-enrichment.

Universal equations to relate 6D values of microbial CH4 to H2O in freshwater environments should be used with caution because they do not adequately express depth-dependent variations in SD-CH4 values. Sporadic emission of gas bubbles from deep soil-layers may release CH4 with SD values that deviate strongly from relationships predicted using laboratory incubations. Such incubations are either dominated by acetate fermentation and do not properly reflect the influence of H20 on SD-CH4 values in older soils [29,30] or have developed unnaturally high concentrations of H2 that may result in abnormally low GD-CH4 values [68].

Acknowledgements

This study was supported by NSERC research grants to FJL and WSF, and generous donations from the EJLB Foundation and the Richard Ivey Foundation. ERCH thanks NSERC, Petro Canada Inc., the Geological Society of America, the Arcangelo Rea Family Foundation and the University of Western Ontario for financial support. We thank Wardens G. Mouland and T. Linke for permitting sampling access to Point Pelee National Park and M. Leonard, formerly of the Upper Thames Conservation Authority, for access to

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the Sifton Bog. The insightful comments of two anonymous reviewers were also very much appreciated.

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