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Chemical Geology 161 Ž1999. 291–314 www.elsevier.


Carbon and hydrogen isotope systematics of bacterial formation and oxidation of methane
Michael J. Whiticar
) School of Earth and Ocean Sciences, UniÕersity of Victoria, P.O. Box 3050, Victoria, BC, Canada V8W 2Y2 Received 30 March 1998; accepted 23 November 1998

Abstract The diagenetic cycling of carbon within recent unconsolidated sediments and soils generally can be followed more effectively by discerning changes in the dissolved constituents of the interstitial fluids, rather than by monitoring changes in the bulk or solid organic components. The major dissolved carbon species in diagenetic settings are represented by the two carbon redox end-members CH 4 and CO 2 . Bacterial uptake by methanogens of either CO 2 or ‘‘preformed’’ reduced carbon substrates such as acetate, methanol or methylated amines can be tracked with the aid of carbon Ž13 Cr12 C. and hydrogen ŽDrH '2 Hr1 H. isotopes. The bacterial reduction of CO 2 to CH 4 is associated with a kinetic isotope effect ŽKIE. for carbon which discriminates against 13 C. This leads to carbon isotope separation between CO 2 and CH 4 Ž ´ C . exceeding 95 and gives rise to d13C CH 4 values as negative as y110‰ vs. PDB. The carbon KIE associated with fermentation of methylated substrates is lower Ž ´ C is ca. 40 to 60, with d13 C CH 4 values of y50‰ to y60‰.. Hydrogen isotope effects during methanogenesis of methylated substrates can lead to deuterium depletions as large as d DCH 4 s y531‰ vs. SMOW, whereas, bacterial DrH discrimination for the CO 2-reduction pathway is significantly less Ž d DCH 4 ca. y170‰ to y250‰.. These field observations have been confirmed by culture experiments with labeled isotopes, although hydrogen isotope exchange and other factors may influence the hydrogen distributions. Bacterial consumption of CH 4 , both aerobic and anaerobic, is also associated with KIEs for C and H isotopes that enrich the residual CH 4 in the heavier isotopes. Carbon fractionation factors related to CH 4 oxidation are generally less than ´ C s 10, although values ) 20 are known. The KIE for hydrogen Ž ´ H . during aerobic and anaerobic CH 4 oxidation is between 95 and 285. The differences in C and H isotope ratios of CH 4 , in combination with the isotope ratios of the coexisting H 2 O and CO 2 pairs, differentiate the various bacterial CH 4 generation and consumption pathways, and elucidate the cycling of labile sedimentary carbon. q 1999 Elsevier Science B.V. All rights reserved.
Keywords: Methane; Carbon dioxide; Carbon isotopes; Hydrogen isotopes; Methanogenesis; Methanotrophy; Bacteria; Soils; Sediments

1. Introduction The interplay between bacterial processes of methanogenesis and methanotrophy continues to at)

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tract the interest of microbiologists and geochemists, despite the fact that CH 4 , even at saturated concentrations in shallow organic-rich sediments, represents only about 1‰ of the total organic carbon present Že.g., 10 m water depth, ca. 4% C org , porosity ca. 0.9.. Apart from the more fundamental interests,

0009-2541r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 5 4 1 Ž 9 9 . 0 0 0 9 2 - 3


M.J. Whiticar r Chemical Geology 161 (1999) 291–314

such as bacterial physiology or organic matter remineralization, some of this curiosity reflects the economic fact that roughly 20% of the worldwide natural gas reservoirs are from bacterial sources ŽRice, 1992; Rice and Claypool, 1981; Whiticar, 1994.. The bacterial CH 4 cycle also receives current attention owing to its impact on the atmospheric CH 4 budget ŽKhalil and Shearer, 1993.. This follows the identification that CH 4 in the atmosphere is: Ž1. a significant radiatively active or ‘‘greenhouse’’ gas, Ž2. represents an important sink for tropospheric hydroxyl radicals, Ž3. increasing in tropospheric concentration at a rate of ca. 18 to 9 ppb yry1 Že.g., Dlugokencky et al., 1995., and that Ž4. bacterial CH 4 is the primary source Žca. 80%–90%. of this atmospheric CH 4 Že.g., Cicerone and Oremland, 1988; Whiticar, 1993.. Furthermore, the processes of methanogenesis and aerobic CH 4 oxidation are also attractive to study by microbiologists because they frequently involve only single or two-carbon reactions. Thus, these bacterial pathways are more easily followed and reproduced in the laboratory. For the geochemist, methanogenesis and methanotrophy are interesting because they are sensitive indicators of the state of diagenesis in a particular environmental setting. The interest expressed in this paper reflects the intricate diagenetic relationships in the carbon cycle that permit the most oxidized form of carbon to reside and be maintained adjacent to its most reduced form ŽFig. 1.. The physical Ždepth. separation of CH 4 formation and consumption is often a transitional feature rather than distinctly zoned. The activity maxima of the respective CH 4 formation and consumption processes can be separated spatially from each other by several decimeters in depth, as in marine sediments, or at a sub-millimeter scale, as in certain stromatolitic hydrothermal or hot spring environments Že.g., Kuhl and Barker Jørgensen, 1992.. An elegant method to track the complimentary processes of methanogenesis and methanotrophy is provided by the stable isotopes of C and H. The partitioning of the light and heavy isotopes of hydrogen and carbon Že.g., Fig. 1. and the resultant isotope signatures can be diagnostic for the identification of methane type and pathway. This paper reviews the stable isotope systematics primarily associated with

Fig. 1. Carbon cycle for bacterial methane formation and consumption. Numbers indicate the carbon and hydrogen isotope fractionation factors Ž ´ C , ´ H . for the various mechanisms Žsee Eq. Ž7... MeOH s methanol, Ac s acetate, TMA s trimethylamine and DMS s dimethylsulphide, DES s diethylsulphide, ET s ethanethiol.

the incorporation and release of C and H during methanogenesis and methylotrophy, i.e., the bacterial formation and consumption of CH 4 .

2. Methanogenic pathways The microbiology, ecology and biochemistry of the various bacterial CH 4 formation pathways have been reviewed extensively in several monographs Že.g., Oremland and Capone, 1988; Zehnder, 1988; Garcia, 1990; Konig, 1992; Marty, 1992 .. ¨ Methanogens, fermentative archaebacteria, are obligate anaerobes that metabolize only in anoxic conditions at redox levels Eh - y200 mV. The fact that

Ž 2. methanogenesis proceeds uninhibited once anaerobic conditions are established ŽFig. acetate and formate. ¨ 1981. can be represented by the general reaction: CO 2 q 8Hqq 8ey™ CH 4 q 2H 2 O. Hydrogeno-methylotrophic and alcoholotrophic methanogens have also been discussed ŽNeue and Roger. In these sulphate-rich environments.e. Competition by the SRB is essentially absent so that the short-chained volatile fatty acid ŽVFA.. The primary methanogenic pathways are referred to as: Ž1. acetotrophic and methylotrophic pathways. the availability of nutrients..1. methane is generally present at only trace concentration levels Ž. hydrolytic and fermenting bacteria. 2. to classify methanogenic pathways uses microbial ecological relationships.. Methanotrophy is discussed in detail later. ultimately to methane. Methanogenesis by competitive substrates is severely restricted in water columns or sediment pore fluids with abundant dissolved sulphate. methanogenesis commences using competitive substrates. SRBs. These competitive and noncompetitive methanogenic pathways are outlined below. termed the ‘‘carbonate reduction’’ pathway. Most of the available labile carbon compounds in these zones are metabolized by nonmethanogens and released.. Ž2. . nitrate or nitrite levels are due primarily to the instability of their F420 –hydrogenase enzyme complex ŽSchonheit et al. Methanogens utilize relatively few and simple compounds to obtain energy and cell carbon: Ž1. In freshwater Žlow sulphate. recognized that four bacterial assemblages operate in this complementary fashion: Ž1. anaerobic CH 4 consumption in the sulphate zone also assists in maintaining the low CH 4 levels observed. or severely restricted to the methanogens.e. Once the available dissolved sulphate is exhausted. in part. and the net reaction for the acetate fermentation pathway is: U CH 3 COOH ™U CH 4 q CO 2 . In sulphate-rich zones. SRB successfully outcompete methanogens for carbon substrates. such as sulphate reducing bacteria ŽSRB. where the U indicates the intact transfer of the methyl position to CH 4 .g.g. 3. Hq-reducing bacteria. It is important to remember that the anaerobic remineralization of organic matter. to the dissolved bicarbonate pool ŽFig. competitive and Ž2. Ž2. A generalized CH 4 distribution for marine sediments with elevated organic carbon content. Conrad Ž1989. Carbonate reduction is the dominant methanogenic pathway under these conditions because of the relatively depleted levels of other methanogenic substrates. alternative to biochemistry. 1985. environments. and the SRB become inactive. in addition to acetate or methylated amines. Conrad et al. hydrogenotrophic.. shallow waters with high depositional rates. The former are those substrates which can be utilized more effectively by other bacterial assemblages. e.. for the available hydrogen. Other factors. and thus are either made unavailable Žout-competed. In addition. 1993. typically those systems with dissolved sulphate greater than 200 m M. pool is available and provides. is presented in Fig. 3. by the SRB and because of the accumulation of a sizable dissolved bicarbonate pool in this sulphatefree zone ŽFig.. Methanogens form methane by pathways that are commonly classified with respect to the type of carbon precursor utilized by them. or in the case of carbonate reduction. i. i. CompetitiÕe substrates Competitive substrates include CO 2 Žreduced by hydrogen. acetotrophic – 19 species described and Ž3. or consumption reactions can determine the occurrence of bacterial methane in a particular environment... such as marine sediments where methanogenesis is severely curtailed.5 mM. Whiticar r Chemical Geology 161 (1999) 291–314 293 they cannot tolerate significant p O 2 .0. including redox potential. non-competitive. substrates and terminal electron acceptors. An approach. Ž3.. hydrogenotrophic – 52 species described... namely methanogenic substrates.J.. methylotrophic – 10 species described Že. such as acetate. The first one.. relies on consortia of bacteria and microbes that successively break down larger molecules.M. methanogenic bacteria. usually at greater sediment depth.. 2. Non-competitive substrates are those which are more suitable to methanogens and less attractive to other bacteria. Ž 1. homoacetogenic bacteria and finally Ž4. 2.

Non-competitiÕe substrates Non-competitive substrates for methanogenesis include both the acetotrophic and methylotrophic pathways that utilize the carbon substrates methanol. Previous investigators stated that acetate fermentation is responsible for roughly 70% of the methanogenesis in freshwater environments. 1992. A typical fermentation reaction for these substrates by methylotrophic methanogens can generally be represented by: CH 3 y A q H 2 O ™ CH 4 q CO 2 q A y H. 2. DMA and TMA.2. Kiene et al. e. Marty. Ž 3. switch over to or start reducing the bicarbonate carbon source with hydrogen.. and the methanogens Žexcepting the obligate methylotrophs. respectively....and tri-methylamines ŽMA. Takai Ž1970. Ž1975.. 2. Daniels et al. e. 2. The utilization of other methylated substrates andror carbonate reduction are suggested as constituting the remainder.. 1984. Similar results have been cited for sewage sludges and from culture studies ŽBryant. Whiticar r Chemical Geology 161 (1999) 291–314 Fig.. methylated amines. di.J. 1986. Winfrey et al.294 M.. Ž1977.. dimethylsulphide ŽDMS.. 1982. important substrates for methanogenesis ŽFig.. Schematic diagram showing methanogenic pathways in marine and terrestrial environments. Methanogenesis by carbonate reduction in freshwater environments is initially less significant. such as mono-.g.. 1979.g. Belyaev et al. and Cappenberg and Jongejan Ž1978. Zehnder et al. Koyama Ž1964. but increases in importance as the other substrate pools become exhausted. .g. and certain organic sulphur compounds. e...

. i.. carbonate reduction.: dx s Ž R a . those environments which are known to be major emission sources of CH 4 to the atmosphere ŽCicerone and Oremland. C and H isotope variations of methane in natural environments The isotope data are reported in the standard d-notation Že.. e. expressed here in permil Ž‰. A s oxic zone. sample y 1 10 3 Ž ‰ . including diethylsulphide ŽDES. B s sulphate reduction zone. Enhanced methanogenesis has also been obtained in culture experiments by additions of various ethylated and sulphide compounds ŽOremland et al. methanethiol ŽMeSH . 3. Whiticar. but they are thought to be less important. The relative importance of some of these methylated substrates as the source for bacterial CH 4 is currently uncertain. C s main methanogenic zone. standard Ž 4.g.J. Their impact as substrates for methanogens is also currently unknown... Ž R a . 1989.. Sediment depth profiles of methane concentration and carbon isotope composition in marine and freshwater environments. 1988b. D s zone of substrate depletion andror shift to alternative substrate for methanogenesis. 1988. However...e. salt marshes and other wetlands. d D. Andreae and Schimel.g.. 1990.M. CH 4 formation utilizing non-competitive substrates is known for environments such as rice paddies. ethanethiol ŽEtSH. . and ethanol ŽEtOH. 3. Whiticar r Chemical Geology 161 (1999) 291–314 295 Fig. d13 C.

the difference in type and magnitude of kinetic isotope effects ŽKIE. The former has a d13 C CH 4 range extending from roughly y50‰ to y20‰ ŽFig. swamp and lacustrine settings..g. The study of C and H isotope systematics based on in vivo observations of methanogenesis have. CD-diagram for classification of bacterial and thermogenic natural gas by the combination of d13 C CH 4 and d DCH 4 information..1. to define compositional fields for the different methanogenic pathways. respectively. but not exclusively. together with those of the coexisting bicarbonate and water species. and to the generally higher temperatures for Fig. including marine. Thermogenic. 4. 4. ratios relative to the PDB and SMOW standards. it was also possible with stable C and H isotopes to differentiate between the different bacterial and thermogenic CH 4 types. generally emphasized the competitive substrates.296 M. 4 due to the isotopic offset caused by the C and H isotope effects associated with the photochemically mediated OH-abstraction reaction Že. 1993.. marsh. 4. 3. methane Thermogenic CH 4 is generally. in the past. Whiticar et al. Using field measurements of CH 4 from various recent and ancient sedimentary environments. The combination of d13 C CH 4 and d DCH 4 values defining the various natural sources of CH 4 in the geosphere is demonstrated in the CD-diagram ŽFig. The dissimilarity in isotope distributions of bacterial and thermogenic CH 4 accumulations is related to several factors including precursor compounds. involved. Whiticar r Chemical Geology 161 (1999) 291–314 where R a is the 13 Cr12 C or DrH Ž'2 Hr1 H. non-bacterial.J. . used the combination of C and H isotope data of CH 4 . Ž1986. The atmospheric methane CD pair does not fall within any of the fields in Fig.. enriched in 13 C compared with bacterial CH 4 . Whiticar.

Bacterial methane The term ‘ bacterial ’ is preferred over ‘ biogenic ’ because the carbon in both bacterial or thermogenic natural gases. the carbon isotope separation between thermogenic CH 4 and organic matter Ž d13 C CH – d13 C C . methane that is formed in Ž1.1. 4. 4 for comparison. 4. 4. it is possible to further classify them according to the source rock type Žkerogen type. These fields are now labeled in Fig...g. kerogen types I and II. are more important in freshwater environments. Whiticar r Chemical Geology 161 (1999) 291–314 297 the thermogenic generation of hydrocarbons. namely. for example. the hydrogen isotope ratios of CH 4 are less useful as an isolated parameter to classify natural gases. Although it is common to measure only the carbon isotopes of CH 4 . 4 org Furthermore. is ultimately derived from or has been part of the biological loop of the exogenic carbon cycle. In the general . such as geothermal and hydrothermal. varying in d13 C CH 4 from y110‰ to y50‰. freshwater and Ž2. 1988.e. These three explanations are expanded in Section 4. involved. 4. Whiticar... kerogen sources. KIEs.. saline sedimentary environments. C 2 –C 4 . However. that carbonate reduction is the dominant methanogenic pathway in marine environments and methyl-type substrates Žacetate. e. in summary the d13 C CH 4 of thermogenic gases becomes progressively enriched in 13 C with increasing maturity. isotope signature of source material and 2.e. The explanation for this overlap is related to the combined effects of: Ž1. This initial distinction was based only on the classification of the depositional environments and did not explicitly consider the methanogenic pathwayŽs. i. if they are used in combination with additional molecular or isotope compositional gas data.. kinetic isotope fractionation by methanogens. Kinetic isotope effects for carbon The distribution of carbon isotopes during uptake and metabolism of carbon compounds by methanogens is controlled primarily by 1. migrationrdiffusion. However. 30‰ to 0‰.g. i.and H-isotope signatures and ranges of other non-bacterial methanes. 3. In certain instances. variations in Cand H-isotope composition of precursor organic matter.2. the d DCH 4 information can be particularly valuable. Factors controlling isotope signatures in bacterial methane 4. including most methane. hydrogen-rich organic matter. coaly Žtype III.. Due to the considerable overlap in d DCH 4 between certain bacterial and thermogenic methane types. 1986. A detailed discussion of isotope variations for thermogenic gases exceeds the framework of this paper Žsee Schoell. methanogenic pathways can also be inferred from the C and H isotope data. Bacterial CH 4 accumulated in marinersaline environments is generally more depleted in 13 C and enriched in deuterium than observed in freshwater environments ŽFig... 1994. In addition to thermogenic methane. Within this large range of values two primary bacterial CH 4 fields were initially identified.. 4 are delineated by compiled CH 4 data from natural environments ŽWhiticar et al. and maturity level from which they were derived. However. The hydrogen isotope ratios of thermogenic CH 4 range from d DCH 4 values of approximately y275‰ to y100‰ ŽFig. 4. In general. Bacterial CH 4 has a wide range of C and H isotope ratios ŽFig. Ž2. The separation of the two bacterial CH 4 fields in the CD-diagram can be made at approximate boundaries of d13 C CH 4 of y60‰ and d DCH 4 of y250‰ ŽFig. mixtures of various pathways andror CH 4 types Že. are presented in Fig. and in d DCH 4 from y400‰ to y150‰. can often generate CH 4 at lower levels of thermal stress and with more negative d13 C CH 4 values than. the CH 4 isotope data have a ‘‘transitional’’ isotope composition that lies between the two fields. Within the range of d13 C CH 4 values known for typical thermogenic gases. the C.M. the hydrogen isotope ratios of CH 4 are particularly helpful in defining the bacterial CH 4 types.. 4 as bacterial methyl-type fermentation and bacterial carbonate reduction.J. and Ž3. This relies on the combination of environmental information with the understanding of microbiological processes presented in Section 2.. respectively. The bacterial fields in the CD-diagram shown in Fig. eventually approaching the 13 Cr12 C of the original organic matter or kerogen Žand rarely even heavier. can vary from ca.

g.g. In a closed system. For larger separations. Ž 6. and R p . methane. and at time t . Substrate–methane Methanol Acetate TMA CO 2 –H 2 DMS DES EtSH 68 to 77 24 to 27 39 55 to 58 44 to 54 49 54 Carbon dioxide–methane 40 to 54 10 49 55 to 58 Fractionation factor Ž ´ C .t s Rr . Hence.: ´ C f 10 3 ln a C f10 3 Ž a C y 1 .. these assumptions can lead to significant errors. e. The isotope separation in d-notation between two compounds. respectively: R r . can be expressed as the isotope separation factor Ž ´ C . x is the isotope ratio of the precursor substrate. e. 12 CH 3 COOH as opposed to 13 CH 3 COOH. the fermentation of acetate involves an enrichment in 12 C on the order of 25‰ to 35‰ for d13 C CH 4 relative to d13 C acetate . and Ž6. acetate or CO 2 . Methanogenesis: culture experiments Substrate Fractionation factors Ž ´ C . where R r.g. 4 is observed for methanogenesis by carbonate reduction. typically less than 25‰ between species. f is the fraction of initial substrate remaining at time t and a is the kinetic isotope fractionation factor. where: 12 Ž 5. t s R r . 13 C-enriched values. 12 CO 2 ™ 12 CH 4 k 13 and 13 CO 2 ™ 13 CH 4 ..J. k Ž 8. Ž7.g.. ŽB. of carbon dioxide–methane 39 to 58 49 to 95 case for each particular compound. a C s13 kr12 k . In these cases. the fractionation factor a C is better defined as the ratio of the reaction rate constants for the two isotopes. that can be approximated by the general forms ŽEqs. d13 C CO 2 and d13 C CH 4 . Marine Žcarbonate reduction. x . Ž5. referring to Eq. Ž1yf .. the continued preferential removal of the isotopically lighter molecules from the carbon pool during methanogenesis results in a progressive shift in the residual substrate towards heavier. This carbon isotope mass balance is commonly described by the distillation functions of Rayleigh Ž1896. Methanogenesis: natural observations Environment Freshwater Žmethylated compounds.g.298 M. f d13 C CO 2 y d13 C CH 4 .. The apparent magnitude of this isotope fractionation varies for different pathways ŽTable 1. there is a corresponding progressive 13 C enrichment in CH 4 that is subsequently formed.. e.. are valid for relatively small isotope separations. and R p. is the isotope ratio of the accumulating product. Ž1. diffuse and react more rapidly and thus are utilized more frequently than the isotopically heavier species.i Ž1yf a . Note that the approximations in Eq. the molecules with the lower isotopic mass Že. common with hydrogen isotopes.. i f Ž ay 1. Ž 7. e. and accumulating initially Ž i . A significantly greater 12 C enrichment Žover 55‰ for d13 C CH relative to d13 C carbon dioxide . .. This discrimination accounts for the strong depletion in 13 C of bacterial CH 4 relative to the precursor substrates. Whiticar r Chemical Geology 161 (1999) 291–314 Table 1 Magnitude of carbon isotope effects associated with methanogenesis ŽA. For example. to describe the isotope ratio of the remaining reactant and accumulated product.

e. aAy B s .M. t s d13 C substrate . Whiticar r Chemical Geology 161 (1999) 291–314 299 For equilibrium isotope situations. Ž d B q 10 3 . where the isotope fractionation occurs during methanogenesis is still uncertain. Ž5. 3 d13 C substrate .J. However. and Ž6.. t s d13 C substrate . this notation has sometimes been used to express the magnitude of the isotope partitioning. . can be converted to d-notation and simplified by the Ž 11 . Ž 10 . Ž 9.. However. . the remaining reactant. The actual locationŽs. at the membrane andror enzymatic level. i q ´ Ž 1 q ln Ž 1 y F . i q ´ ln Ž 1 y F . Theoretical shifts in d13 C CH 4 and d DCH 4 as a result of isotope variations in original organic matter and through secondary effects Žsubstrate depletion and methane oxidation. These Rayleigh distillation functions expressions ŽEqs. the consequence of this fractionation is that within a specific environment a considerable Fig. where: Ž dA q 10 . Ž7.. and taking F s 1 y f . and for the instantaneous product formed: d13 C methane . 5. Normally. isotope equilibrium cannot be assumed for these kinetic reactions. the relationship between a and d is defined as: substitution with ´ ŽEq.. .g. ..

a change in the enzymatic kinetic reaction rates for 13 C vs. Although such variations in the d13 C CH 4 values exist.e. Their culture experiments of methanogenesis by carbonate reduction with Methanococcales at 35–858C gave ´ C values of 76 to 47. Methanosarcina barkeri Thermoautotrophicum Hyperthermophil Temperature Ž8C..2.. Later. and Jedrysek Ž1995. Ž1979. Botz et al. Iversen Table 2 Effect of temperature on the magnitude of carbon isotope effects associated with methanogenesis by carbonate reduction Locationrbacterium Bransfield Strait Ž 3. one could expect a decrease in the isotope effect at higher temperatures. Whiticar. also documented a temperature dependence of isotope fractionation during biological methanogenesis. the magnitude of separation is unlikely to be caused by a change in the isotope effect. as expected from thermodynamic considerations. Temperature effects on carbon isotope Õalues The effect of temperature on the fractionation of carbon isotopes associated with methanogenesis is poorly understood. Ž1979. showed that there is a general trend to lower fractionations with increasing temperature. the isotopic partition of d13 C CO 2 – d13 C CH 4 decreases from 100‰ to 40‰. . Thus.. demonstrated a 2.300 M. Whiticar et al. i. However. they are most commonly observed in methanogenic culture experiments. it has been suggested that the carbon isotope fractionation is dependent on other factors such as substrate concentration and rates of methanogenesis. Fuchs et al. reported for culture experiments that the magnitude of carbon isotope fractionation during methanogenesis by carbonate reduction was dependent on the CO 2 gassing rate. Ž1996. Muller. Whiticar r Chemical Geology 161 (1999) 291–314 range in carbon isotope values can be observed which is dependent on the degree to which the substrate has been depleted ŽFig. 4. stated that the magnitude of the carbon isotope fractionation is also dependent on the rate of methanogenesis by a variety of methanogens and substrates ŽCO 2 and methylated. Martens et al. Over a 1108C temperature rise. they reported that the temperature could influence the rates and pathways of methanogenesis ŽSchutz ¨ and Conrad. indicates. Zyakun Ž1992. demonstrated a similar result. but decreased dramatically as the amount of CO 2 available dropped. Ž8.. the carbon isotope values of bacterial CH 4 can approach those of the original organic matter.. albeit incomplete. that the KIE decreases significantly with increasing temperature.J. test is provided by comparing the a C between CO 2 and CH 4 from carbonate reducing methanogenesis at various temperatures ŽTable 2. but it is not known if this would affect the KIE. equivalent to an ´ C change of ca. 5. This relationship.3 20 37 65 110 ´ C CO 2 –CH 4 86 to 95 77 58 34 to 40 40 References Whiticar and Suess Ž1990. Zyakun Ž1992. Blaut Žunpublished data. In addition to temperature. Increased levels of methanogenesis at higher temperatures is well documented. 12 C ŽEq. It is clear from these experiments that the isotope separation between the precursor and methane decreases with the degree of substrate utilization. Ž7. y1. In addition.5. Above a critical CO 2 threshold the CO 2 –CH 4 isotope separation was constant. In cases of extensive substrate exhaustion. A rough. Ž1986. 86 to 39 for CO 2 –CH 4 pair ŽEq...5-fold increase in CH 4 generation due to a 108C temperature rise. Stetter. It is more likely that the decrease in precursor-methane isotope separation is due to a ‘reservoir effect’ caused by substrate depletion andror diminished rates of substrate transport across the membrane walls of the methanogens. For example. shown in Fig... the methane can in fact be more enriched in 13 C than the starting precursor material. Conrad and Schutz ¨ Ž1988. respectively. 6. 1996. Schutz ¨ et al. Whiticar. Ž1986. showed that diurnal variations in temperature could influence the carbon isotopes.. ¨ Fuchs et al. In some instances where mass balance is not maintained. Huber Žunpublished data. 1997. the maximum expression of the carbon isotope effect corresponds to the situations with high substrate levels. such as loss of the methane initially formed. but again this is probably a methanogenic rate effect and not enzymatic. Empirically.

. subsequently clarified that temperature indirectly exerted some influence on substrate availability due to enhanced microbial remineralization rates.. challenged this and in reply Hornibrook et al. but the magnitude may be dependent on it. which may be operating in the same location. This hydrogen isotope partitioning is present regardless of the methanogenic pathway.. albeit small. demonstrated that shifts to different methanogenic substrates or isotope signatures might also be controlled by seasonal conditions. such as the succession to carbonate reduction after the acetate pool is exhausted. Ž1998. variations in isotope composition of the precursor organic matter. 5. 13 C. Martens et al. In addition. but can also involve admixtures of thermogenic gas. 1997.5. 1996. found that the temperature may have some effect on the rate of methane oxidation but the results were inconclusive.. Ž1986. the form of methanogenesis can shift from one substrate to another. Temperature dependence of carbon isotope partitioning Ž ´ C between d13 C CO 2 and d13 C CH 4 .4. Isotope Õariations in precursor organic matter 4. This mixing is not necessarily restricted to bacterial gas. As expected by the larger mass difference between 1 H and 2 H than 12 C vs. ŽWhiticar et al.3. equilibrium mechanisms dominate the hydrogen isotope distributions in methane. . Whiticar r Chemical Geology 161 (1999) 291–314 301 resolved to what extent kinetic vs. Burke et al. Ž1988b. 1988a. Hydrogen isotope effects Our understanding of the hydrogen isotope effects associated with methanogenesis is limited. complicate the situation. For example. Ž1996. could be anticipated. to influence the methanogenic pathway and the carbon and hydrogen isotope signatures. 4. As discussed below.. by methanogens via the carbonate reduction pathway. This could be the result of contributions of CH 4 from different methanogenic pathways ŽFig.. 4. Mixtures Bacterial CH 4 with ‘‘mixed’’ or ‘‘transitional’’ isotope signatures can often be present in a gas sample. 1993. or hydrogen isotope exchange Žde Graaf et al. Hornibrook et al. 5. it remains unIt is more difficult to assess the third point. the hydrogen isotope partitioning is larger than carbon isotopes.. 1986. With the exception of dissolved ÝCO 2 Žsee below. Ž1997. These gas mixtures could also be the result of physical mixing of different CH 4 pools in response to vertical or lateral migration or diffusion of gas. Ž1992.g. The magnitude to which the C and H isotope compositions of the various methanogenic substrates deviate from those of the bulk organic matter is not well known. namely. although some effect. Waldron et al. Temperature was suggested by Hornibrook et al. isotope measurements are sparse for specific methanogenic substrates in natural environments. 6.. There is no evidence indicating that temperature affects the carbon or hydrogen isotopes related to methane oxidation. there are suggestions that factors such as hydrogen concentration ŽBurke. Burke et al. C and H isotope values of bulk organic matter that are available for specific environments exhibit the approximate isotope ranges shown in Fig. 1992. nor is the magnitude of any intramolecular isotope fractionation present in Fig. either concurrently or successively..J. Work in shallow marine sediments by Sansone and Martens Ž1981.. This is observed in some freshwater systems Že.M. and Kelley et al. Ž1998. Empirical evidence suggests that substantial enrichments in 1 H do occur as hydrogen is transferred to the methane molecule from either organic precursors or formation water.

. it has been assumed up to now that the natural C and H isotope signatures of a substrate in a specific setting are similar to those of the bulk organic matter. Methylated substrates available for fermentation by methanogens often are present only in minor amounts. Meinschein et al.. Bransfield Strait. carbon isotopes of methane and bicarbonate in the marine sediments from core 1327. bicarbonate and sulphate and Žb. Antarctic. Whiticar et al. However. Concentration depth profiles of methane. 1974.. Analytical techniques now are available to measure C and H isotope ratios in methane and the precursors in the picomole range Že.b. Fig. Although this may control the rate of methanogenesis via methyl-type fermentation. This is due to an effective combination of formation and utilization that leads to a relatively short residence time. . Whiticar r Chemical Geology 161 (1999) 291–314 such substrates well understood ŽYankwich and Promislov.. provided that severe substrate depletion has not occurred and that large interor intramolecular variations are not present.. 1991.. that should help resolve this issue. Ža. 1953. 1998a.302 M. Rossmann et al.J. 7.g.

Thus. with values most commonly around 65 to 75 ŽFig. due to the preferential uptake of carbon over nitrogen. the isotope separation between CH 4 and CO 2 remains consistent for.e. In this case. 4.g. Isotope systematics of methanogenesis with the coexisting CO 2 and water 5. Typically only determinations of bulk d D C are o rg available for natural environments without the respective DrH values for specific substrates or hydrogen isotope distributions within the precursor molecules... 1981. indicates that CO 2 is continually added by organic remineralization to the pool in the methanogenic zone.. The ambiguity in determining the methanogenic pathway can sometimes be resolved by considering the isotope information of CO 2 Ž d13 C CO 2 . In the case of carbon. This strict relationship between d13 C CO 2 and d13 C CH 4 provides important information about methanogenic pathways. 7a.. Similarly. can be used to differentiate the major CH 4 sources. provided one of the first models using gas and interstitial data from sediment cores of the Deep Sea Drilling Program ŽDSDP. the various secondary effects discussed above can obscure the signature of sourceŽs.and intramolecular isotope distributions and effects for hydrogen in organic matter are much less well constrained than for car- bon. the environments where methanogenesis operates with mixed substrates can also be recognized using the CH 4 –CO 2 pairs despite large variations in the actual d13 C CH 4 values. those dominated by fermentation of methylated substrates.1. under certain conditions.. ODP and other sediment cores is illustrated by the 7. which also becomes heavier with sediment depth in the methanogenic zone as predicted by the Rayleigh function ŽEq. CH 4 concentrations first exceed 100 ppb Žweight of gasrweight of wet sediment. ODP. and formation water Ž d D H 2 O . 7b. The carbon isotope separation between CO 2 and CH 4 in the CH 4 zone of core 1327 is consistently around 80‰. 1990. the particular methanogenic pathway. are distinctively lower with ´ C values typically ranging between 40 and 55 ŽFig. the isotope separation factor between d13 C CO 2 and d13 C CH 4 Ž ´ C . and indicative of.J.M.. although the d13 C CH 4 can shift dramatically in response to the changing isotope ratio of the depleting substrate.. later Ocean Drilling Program. These amounts permit more readily the modeling of carbon isotope systematics for methanogenesis by carbonate reduction. Sansone and Martens. 1992. which coexist with the CH 4 . 1986. This change was accompanied by a sharp decrease in molar C:N ratio Žcarbonate alkalinity:ammonia. The fact that the CO 2 concentration does not substantially decrease in this zone.g. Carbon isotopes Although the CD-diagram ŽFig.5-m piston core taken at station 1327 in the Bransfield Strait. 8.. Blair and Carter. A key point is that this carbon isotope fractionation factor tends to remain more or less constant for each specific setting or diagenetic environment.. This depletion is in turn expressed in the d13 C of the CH 4 . i. Ž7. such as organic acids. The variations in inter.. beneath the sulphate reduction zone ŽFig. e.. 8 by the production arrow. the corresponding fractionation factors for methanogenesis in freshwater environments. Whiticar r Chemical Geology 161 (1999) 291–314 303 the amount of methane produced can exceed saturation ŽFig. In comparison. Antarctic ŽWhiticar and Suess. In natural marine or saline environments ´ C associated with methanogenesis predominantly by carbonate reduction ranges from 49 to over 100.. makes it difficult to isotopically model their removal Že. This is illustrated in Fig. 3. 5. The continual removal of dissolved bicarbonate by methanogens is marked by the reduction in the increase of dissolved ÝCO 2 concentration with depth from the sulphate reduction zone to the zone of methanogenesis. organic-rich marine sediments can have substantial bicarbonate concentrations Ž) 100 mM.. although overall there is a 12 C depletion by the methanogens. after Whiticar et al. An illustration of results similar to those found in numerous DSDP. in the corresponding pore fluid or water mass. Claypool Ž1974. In contrast.. . The more rapid uptake and utilization of 12 C bicarbonate by methanogens in core 1327 leads to a gradual shift in d13 C CO 2 towards heavier values in the older sediments ŽFig. is defined by Eq. Ž15. Some culture experiments have used additions of water and substrates with known hydrogen isotope values to assess the methanogenic processes and magnitudes of the related isotope effects. as is developed in greater detail below. 8.. The low concentration of methanogenic substrates..

Ž1974.. Similar to that reported by Rosenfeld and Silvermann Ž1959.J.. Ž1986. 8. found ´ C to be ca. 55 for carbonate reduction in their batch cultures. and Daniels et al. the largest CO 2 –CH 4 separation was observed for CO 2-reduction. ´ C s 58 ŽTable 1. Methanogenesis by carbonate reduction Žsaline. The carbon isotope fractionation factors between CO 2 and CH 4 . the coexisting formation water is essentially the initial hydrogen source Ž100%. has a larger CO 2 –CH 4 isotope separation than by methyl-type fermentation Žfreshwater region.2. i. 1988a. The fermentation of methylated substrates is thought to involve an intact transfer of the three methyl hydro- .. For comparison. 24 for acetate fermentation.304 M. and Whiticar et al. the authors reported ´ C to be ca. Table 1 also provides the carbon fractionation factors between CH 4 and various substrates Žafter Oremland et al. Predictably. Fuchs et al. This has been confirmed experimentally by Pine and Barker Ž1956. is due to methanogens deriving a specific proportion of their hydrogen for CH 4 formation ultimately from the water in which they live.e. The figure also shows the observed CO 2 –CH 4 carbon isotope partitioning trajectories resulting from both bacterial methane formation and oxidation processes. Ž7.. For carbonate reduction.b.. Whiticar r Chemical Geology 161 (1999) 291–314 Fig. associated with methanogenesis in marine and freshwater environments. as determined by culture experiments. This is comparable to the carbon isotope fractionation value of ´ C ca. Ž1984... Similarly. Waldron et al. Ž1979.. marine region.. Hydrogen isotopes The relationship between the hydrogen isotope ratios of bacterial CH 4 and the coexisting formation water can also be employed to track methanogenic pathways. introduced by Nakai et al. methanol exhibits the largest carbon isotope fractionation between a methylated substrate and the methane formed. Ž1987. ´ C me thanol – methane s 68 to 77 ŽTable 1.. Ž1998. 1994. are summarized in Table 1.. 57 reported by Zyakun Ž1992. for carbonate reduction culture with CO 2 concentration as above and independent of the critical threshold. according to Eq. Combination plot of d13 C CH 4 and d13 C CO 2 with isotope fractionation lines Ž ´ C . and Krzycki et al. In the same paper. 5. This consideration. Ž1980. Woltemate et al. and developed by Schoell Ž1980. or methane consumption. Whiticar..

Ž 12 . are dependent on the isotope effects involved in the abstraction and transfer of the hydrogen. Ž7. This intact hydrogen transfer has been demonstrated in culture studies with deuterated substrates ŽPine and Barker. to the CH 4 molecule ŽFig. Based on the current SEOS ŽSchool of Earth and Ocean Sciences. 1995. and BGR ŽBundesanstalt fur ¨ Geowissenschaften und Rohstoffe. Ž13. Hannover. can be modified to: d DCH 4 s f Ž d D H 2 O y 160 . the magnitude of the isotope offset is site specific. most studies suggest that b associated with methanogenesis by methyl-type fermentation is significantly larger than for carbonate reduction. However. is the dissociation of water to hydrogen gas either inorganically or via biological processes. e.. Fig. There is probably not a universal value for b with respect to methylated substrates. Whiticar et al. they are also additionally dependent on the hydrogen isotope ratio of the methyl hydrogens. Sources of hydrogen incorporated into methane through the carbonate reduction and methyl-type fermentation methanogenic pathways. y80‰. The empirical relationship expected between d DCH 4 and the coexisting d D H 2 O as depicted in Fig. Žd. d DCH 4 and d D H 2 O are readily measured... Sugimoto and Wada Ž1993. These latter culture values contrast strongly with those known empirically for natural . Ž12. where f is the relative proportion of methanogenesis by carbonate reduction Ž f s 0 to 1. qŽ 1 y f .. 10. they also calculated m and b values of 0.. the situation is more complicated.683 and 317 for the carbonate reduction pathway ŽL6 in Fig. is the incorporation of hydrogen gas as the final hydrogen into the methane molecule Žthe magnitude of the isotope effect is poorly constrained. of 160. in the isotope mass balance..J. but our present deficiencies in precise information on the distribution of hydrogen isotopes in methylated substances make solution of Eq. 1998a. is the direct synthesis of hydrogen gas by bacterial activity. Interestingly. Eq.. respectively. with an associated isotope effect Ž ´ H .. is a potential direct utilization of water by methanogens during fermentation. . In methanogenic environments with more than one active pathway. Daniels et al.437 and 302 ŽL4 in Fig. The letters refer to pathways: Ža. 1980. to fermentation by methylated substrates. In recent years..M. However. Whiticar r Chemical Geology 161 (1999) 291–314 305 gens Ž75%. ranges on the parameters can be estimated.b. Žb. values of b known for methylated substrates are greater than 300 and extend up to 377. 10. as shown in Fig. y b . is the direct transfer of methyl–hydrogen from the organic matter Ž d Dorg ca.g. Ž 13 . e. Že. difficult. Žf. The combined hydrogen isotope effects during carbonate reduction in various environments appear to be remarkably consistent around 160‰ to 180‰ for CH 4 relative to the formation water. is the direct utilization of water–hydrogen during carbonate reduction. investigated the relationships between d DCH 4 and d D H 2 O for rice paddy soil during methanogenesis in culture experiments. where m s 1 holds for carbonate reduction. To accommodate the mixture of two pathways. Victoria. 1956.25d D hydrogen . The values for ‘ b ’ in Eq. 9.25 is expected for the fermentation of methylated substrates. Ž12. Ž 0. 9 should be: d DCH 4 s m Ž d D H 2 O . Žc. combination of carbonate reduction and fermentation of methylated substrates. q Ž 0. 10. For the methylated compounds. 9. d Dmethyl is the hydrogen isotope ratios of the intact-transferred methyl group and d D hydrogen is that of the final hydrogen incorporated.g. and m s 0. 1957. Rather. the hydrogen isotope systematics associated with methanogenesis have been revisited.75d Dmethyl . for methyl-type fermentation. Although they only indirectly calculated the relative importance of the two methanogenic pathways. Pine and Vishniac. data bases. the authors reported values for m and b according to Eq.. acetoclasts. of 0.

as a function of the hydrogen isotope ratio of the coexisting formation water Že. if one assumes d Dorg value of y120‰ for the three methyl hydrogens donated. Ž12. for freshwater Ž F : R s 80:20. gave d D H 2 O – d DCH 4 values similar to methyl-type fermentation ŽFig. Ž1986. and carbonate reduction ŽL6..25. for methyl-type fermentation ŽL4. after Eqs. Sugimoto and Wada Ž1995. 10. and of Waldron et al. L3 shows the typical Ž d D H 2 O – d DCH 4 relationship observed by Whiticar et al. are shown as points 7–9. and Ž13. Ž0. Certainly some of the methanes strongly depleted in deuterium. found d D H 2 O – d DCH 4 relationships indicating mixed reactions ŽFig.. Ž1988a.. F : R s 100:0. the high H 2 concentrations in the culture experiments of Sugimoto and Wada Ž1995. For example. point 9. 160‰ ŽL1. Burke et al.25 and an offset b of 300‰ to 370‰ ŽL2..g. Ž12. Methanogenesis by carbonate reduction follows a slope ‘‘ m’’ of 1 with an isotope offset ‘‘ b ’’ of ca.. 321. and those in ŽWhiticar et al. Additional data of Balabane et al..306 M.. F : R s 0:100. They countered that in comparison with natural settings.. The explanation offered by Sugimoto and Wada Ž1995.. reported that their natural wetland studies fit the model of Whiticar et al. The methyl-type fermentation pathway has a slope m of ca. and a d DCH 4 of y400‰..e. suggested that hydrogen isotope exchange could explain the difference in m and b values ŽEq. this would . are difficult to explain by direct methyl hydrogen incorporation. those lighter than d DCH 4 ca. 0. y400‰. Hornibrook et al. The relationships of Sugimoto and Wada Ž1995. water column..J. 10. 1986.. Ž1988a. for methyl fermentation between their results Ž0.437 and 302. Dependence of hydrogen isotope ratio in methane Ž d DCH 4 .. 10. Burke et al. Ž1998. Whiticar r Chemical Geology 161 (1999) 291–314 Fig... Ž1987. for a mixture of the two pathways ŽL5. environments. A similar effect was proposed by Burke Ž1993. whereas the culture study of Balabane et al. respectively. Ž1996. points 7 and 8. may have affected the magnitude of the hydrogen isotope partitioning and led to the observed discrepancies. porewater. Ž1997. Ž1986. i. Ž1989. for the difference in the carbonate reduction values between freshwater and marine environments was that the former has higher hydrogen concentration and longer hydrogen residence time. Ž1989. and Grossman et al. and Grossman et al. Ž1987. are shown as dashed lines. Support for hydrogen isotope exchange during acetate fermentation comes from the work of de Graaf et al.

. They calculated the magnitude of this isotope fraction Ž a H 2 O – CH 4 .g. the flux of CH 4 into the atmosphere could be orders of magnitude greater. Whiticar. In the simplest of terms. Whiticar and Faber. Ž1988. Certainly. Steudler et al. but the arguments presented for anaerobic oxidation from a geochemical perspective are convincing ŽIversen and Blackburn. numerous questions require de- tailed attention. Bacterial CH 4 consumption can be by either aerobic or anaerobic oxidation. Alperin et al. The former is a common process in freshwater settings normally at the anoxicroxic interface. 3. 1986.J. Comparison of hydrogen isotope ratio in organic matter Ž d Dorg . The processes controlling the distribution of hydrogen in methane during methanogenesis remain unclear. Conrad. 1989. 1989. The lines are the mass balance for the various values of the resultant d DCH 4. the dramatic decrease in CH 4 concentrations across the CH 4 production–sulphate reduction boundary ŽFig. Ž13. for example within soils or within the uppermost centimeters of sediments in swamps or lakes Že. Anaerobic CH 4 consumption was controversial for a long period amongst microbial ecologists.. 1975.. . as a function of the hydrogen isotope ratio of the final hydrogen attached during methyl-type fermentation Žafter Eqs. 1988. Whiticar r Chemical Geology 161 (1999) 291–314 307 give by Eq. The strongest geochemical indications for anaerobic CH 4 oxidation are: Ž1.47. suggested that the hydrogen isotope signature in bacterial methane is set by the enzymatic incorporation of hydrogen from water and not by the precursor hydrogen on the methyl group. Claypool and Kaplan. including that of possible hydrogen isotope exchange. and Ž13. found values for m and b ŽEq. the concave downward shape of the depth Fig.M.. to be 1. Bossard.. 1993.g. Over the past 20 years studies of CH 4 distributions in marine sediments identified the diagenetic zonation between sulphate reduction and CH 4 formation Že.. 1977. 1981... Ž12... 1985. and Ž3. This indicates that either the hydrogen in the precursor methyl group is more depleted in deuterium than bulk organic matter or some isotopic exchange has occurred. Without this hydrocarbon metabolism. 6. 11. of 0. and the translation of results from culture experiments to natural environments.623 and 319 for cultures of landfill material with both methanogenic pathways operative at unknown relative proportions ŽL5 in Fig. Rudd and Hamilton. 1981. They proposed that this enzymatic control is operative for both acetoclastic and carbonate reduction methanogenic pathways. the systematic shift in C and H isotope ratios of CH 4 consistent with the loss of CH 4 in the consumption zone Že. 1974. Martens and Berner. Waldron et al. rates of methanogenesis. Ž12. Waldron et al.... Ž1988. a non-sensical d D H value of y1240‰ for the final hydrogen ŽFig. effects of hydrogen partial pressures. Isotope effects associated with bacterial methane oxidation Bacterial consumption is a major sink of CH 4 in the geosphere and in water columns... Ž2.g. the preferential loss of CH 4 compared with higher molecular weight hydrocarbons in the same location. Iversen and Jørgensen. This methanotrophic process is particularly active in marine and brackish sediments. 11.. 1982. 10. normally at the base of the sulphate reduction zone. and its impact on the global atmospheric thermal and chemical budgets would be even more significant ŽWhiticar.

Bacterial consumption of CH 4 appears to proceed at a significantly greater rate than for higher molecular weight hydrocarbon gases such as ethane. 50 Ž j. Lines A and j. the concave upward shape of the CH 4 concentration profile could not be maintained by diffusion and advection mechanisms alone Žsee Reeburgh. Whiticar and Faber Ž1986. 12. or Ž3. 1978.J. Whiticar r Chemical Geology 161 (1999) 291–314 profile of dissolved sulphate concentration ŽFigs. approximately 0. Ž2. restricted in situ methanogenesis. combining the molecular and isotope compositional ˇ are calculated mixing lines for possible gas bacterial and thermogenic mixtures with end-member isotope information. that specific nutrients or co-metabolites are not present. Vertical gas ebullition could rapidly introduce CH 4 into the sulphate zone.. 10 5 . y45‰. and y55‰. 4 The relative compositional effects of migration or oxidation are also indicated. Methane buildup in the sulphate zone may be related to consumption rates being lower than the influx of CH 4 . propane Fig. However. microbial uptake by SRB and vertical pore fluid advection. Possible explanations for this are: Ž1. and molecular ÄC 1rŽC 2 q C 3 . Analogously.. . Ž d13 C CH . and Adams and van Eck Ž1988. ˇ . Bacterial anaerobic oxidation at the base of the sulphate zone is responsible for active CH 4 removal. diagram Žafter Bernard et al. Oremland et al. demonstrated that CH 4 can persist in the presence of trace amounts of sulphate.. Natural gas interpretative Ž‘‘Bernard’’. or in certain circumstances the buildup could be due to inhibition of CH 4 oxidation. Martens and Berner. in these environments there is a CH 4 concentration threshold below which CH 4 is not attractive to methanotrophs.4 compositions of y100‰. and other investigators.. is the result of the balance between downward diffusing sulphate. 1977. 3 and 7a. complete consumption of CH 4 does not occur and trace levels of CH 4 are found in both sulphate-bearing and oxic environments. y45‰. 1976.. respectively. 5000. 2 ŽA.2 mM in the sulphate zone of marine. brackish and freshwater sediments. Ž1987.308 M. including Martens and Berner Ž1977.

as CH 4 is removed by bacterial oxidation the residual gas is continually enriched in Table 3 Magnitude of C and H isotope effects associated with methane consumption ŽA. 1968 Lebedew et al. has been observed for the hydrogen isotopes in CH 4 . This fractionation is most pronounced for the CH 4 carbon isotopes. and in some cases. Methanotrophy: laboratory experiments Aerobic cultures Silverman and Oyama. in an aerobic culture study lay between 95 and 285 ŽColeman et al. 1990. The latter were also able to model the hydrogen KIEs for CH 4 oxidation at the same location. The first model Žresidual methane. uses the relationship of d13 C CH 4 to the CH 4 concentration in interstitial fluids. To account for variations of the initial CH 4 concentrations between different environments. Larger values of ´ C around 20. of about 4. and isotope composition Ž d C s d13 C CH 4 .i q ´ ln Ž 1 y F .. In CH 4 formation zones which are devoid of thermogenic hydrocarbons. where d13 C CH 4. The second model ŽC 2q enrichment. The shift in d13 C CH 4 to more positive values as a function of CH 4 consumption is shown in Fig. ratio can decrease to values of less than 10.M. a molecular fractionation occurs which relatively enriches the residual hydrocarbon gas in the higher homologues. The only determinations of the hydrogen isotope fractionation factor for CH 4 oxidation Ž ´ H . C 1rŽC 2 q C 3 . 1988 Concentration King et al. 1981 Coleman et al.. The proportion of CH 4 in a hydrocarbon gas mixture can be expressed on a volume percentage basis by the ‘‘Bernard parameter’’. in the main CH 4 formation zone. the initial CH 4 concentration pool is normalized to 100%. Ž5. The best fit to the data was obtained with a carbon fractionation factor Ž ´ C ...g. Three models were developed by Whiticar and Faber Ž1986.i is the carbon isotope ratio for the initial methane pool prior to oxidation Že. 1969 Zyakun et al.. 1986 Residual methane C 2q enrichment CH 4 –CO 2 Alperin et al. methane–carbon dioxide partition models. These models are termed: Ž1. 12 so that CH 4 oxidation can lead to difficulties in the interpretation of natural gas sources. Ž 14 .. The magnitude of the carbon KIE during CH 4 consumption is much lower than that associated with substrate uptake by methanogens. 1989 Tyler et al.. . The fractionated CH 4 uptake is described by a closedsystem Rayleigh function similar to Eq. 1978. 14.. Analogous to methanogenesis.. 1994 Steudler and Whiticar. Molecular ratios less than 50 are also typical for thermogenic hydrocarbon gases. residual methane. of the bacterial gas during CH 4 consump- . a concentration gradient model. 1998 ´ C-methane 4 18 to 14 5 to 20 9 16 to 27 22 up to 5 ´ D-methane 148 ŽB.. do not describe the system satisfactorily.t s d13 C CH 4 .. Culture experiments and models have been used to substantiate this Žcompare Tables 1 and 3. 1981..J. in culture experiments varies between 5 and 30 ŽTable 3. was published by Alperin et al.. 1984 ´ C-methane 11 16 30 5 to 31 13 to 25 7 to 27 ´ D-methane 95 to 285 the heavier carbon isotope fraction ŽFig.. 13. is typically 10 3 to 10 5. Methanotrophy: natural observations Model names Whiticar and Faber. as shown in Fig. d13 C CH 4. as CH 4 is continually removed. Whiticar r Chemical Geology 161 (1999) 291–314 309 or butanes. uses the simultaneous change in both the molecular ŽB s C 1rŽC 2 q C 3 . Ž2. Ž1988.. In the CH 4 consumption zone the C 1rŽC 2 q C 3 .t is that at time t. Several investigators have demonstrated with aerobic culture studies that 12 C–CH 4 is preferentially removed during its oxidative consumption. 1981 Zyakun et al. Thus. chosen to model the data.. Whiticar. to calculate the carbon isotope fractionation of CH 4 during its anaerobic consumption. The magnitude of the carbon isotope fractionation Ž ´ C . 1979 Barker and Fritz. C 1rŽC 2 q C 3 .. C 2q enrichment and Ž3. A fourth.: d13 C CH 4 . the bacterial uptake of CH 4 is associated with a KIE that enriches the residual CH 4 in the heavier isotope. Žsee Bernard et al.

a significant shift in the methane carbon isotope ratio is first seen when most Žca. Ž14. gave lower ´ C values between 1. the carbon isotope separation between CH 4 and CO 2 ŽEq..8 and 5.. CH 4 oxidation causes a clear. Relationship of d13 C CH 4 to the percent Ž%. for these samples is not clear. As described in Section 5. is fairly constant and indicative of the formation pathway Žmethane–carbon dioxide partition model.. 13.48C. The carbon isotope fractionation factors vary from ´ C s 7 to 14. as indicated in Fig. In contrast. The explanation for the lower bacterial selectivity Fig. of the methane has been oxidized. From the d13 C CO 2 . Initially.. residual methane from bacterial methane consumption ŽEq. according to ´C s ln B t y ln B i Ž ln B t y ln B i . 15 Ža modified version of Fig. determined the isotope shifts in CH 4 from anaerobic Skan Bay sediments as a function of the CH 4 concentration gradient Žconcentration gradient model. The residual methane is the percentage of methane remaining compared with the methane concentration in the main zone of methanogenesis Ž100%. marsh and brackish sediments are shown in Fig.. r10 3 q Ž d C t y d C i . The hydrogen isotope fractionation factor Ž ´ D . 14. Whiticar r Chemical Geology 161 (1999) 291–314 Fig.J. the carbon isotope separation is observed to have ´ C values between 5 and 25. d13 C CH 4 coexisting pairs it is not only possible to distinguish between the methanogenic pathways but also to evaluate CH 4 consumption ŽFig. where the subscripts i and t are for the initial and temporal conditions. 8. By subtracting the possible effects of isotope fractionation associated with diffusion. for derivation of Eq.. which is consistent tion to calculate the isotope fractionation Ž ´ C .. 8. 1990. The compositional shifts for a variety of marine. The carbon isotope fractionation factor ´ C was found to be 8. towards the latter stages of consumption. Residual methane model showing the shift in d13 C CH 4 to more positive values with decreasing methane concentration due to methane oxidation at the base of the sulphate-reducing zone Žlabeled CH 4 Oxidation Zone.. as indicated in the figure. but it may be related to the cold sediment temperatures Žy1. Ž10..310 M. Other data from the Antarctic ŽWhiticar and Suess.. Due to the low isotope fractionation factor Ž ´ C s 4.. respectively Žsee Whiticar and Faber Ž1986.. However. they were able to apply diagenetic equations to estimate the magnitude of both the C and H isotope effects for anaerobic CH 4 oxidation. Ž7. strong decrease in the carbon isotope separation Ž d13 C CO 2 – d13 C CH 4 .. Ž1988. q 10 3 Ž 15 . Alperin et al. the greater amounts of CO 2 than CH 4 typically present obscure the oxidation trend. . for the same samples was 146. 80%. 12.

. V. Iversen... Marburg have led to much of the understanding expressed in this paper. as a radiatively active atmospheric trace gas having direct effect on global climate change. Ž1984. Steudler et al. Univ. In these cases. are excellent tracers of the processes of bacterial CH 4 formation and consumption. Stetter.CH 4 values around 16 to 27. ¨ Gottingen. In view of the increasing atmospheric CH 4 concentration Žca. Ž1989. Ž1989. Ž1981.4 and methane carbon isotope Ž d13 C CH 4 . The magnitudes of the KIEs are consistent and sufficiently large to generate significant isotope fractionations that are diagnostic for the various processes. The carbon KIEs associated with the soil uptake of methane have been reported by King et al. 1993. The latter. The possibility of aerobic methane consumption in soils was recognized by Keller et al. The range of C and H isotope effects from experimental and field observations are summarized in Table 3. and.5%–1% annually in the lower troposphere. ŽTable 3.g.. it is important to attach precise isotope signatures to the various CH 4 sources. methanotrophs appear to be less selective between the lighter and heavier carbon isotopes than the methanogens. with the values reported for aerobic cultures by Coleman et al. Ž1994.. showing the combined shifts in molecular ÄC 1 rŽC 2 qC 3 . USGS. In this way our comprehension of geochemical and microbiological processes involved in natural gas habitats and compositions within the geosphere are the key to understanding atmospheric CH 4 change. Muller and M. ratios resulting from fractionated bacterial consumption of methane in marine and brackish sediments. Steudler et al. This work has been funded. 7. including R. Situations. 1978. 15.M. Conclusions In combination.. Ž15. Whiticar r Chemical Geology 161 (1999) 291–314 311 obic sediments is less than in aerobic culture studies. 0. The former two.J. it can be commented that carbon isotope fractionation during CH 4 oxidation in anaer- . lie between 7 and 14.. Fig. Oremland. Ž1983. Blaut. Tyler et al. K. Stable isotopes are an elegant method to differentiate the various fluxes and numerically constrain the magnitudes of both CH 4 emissions to and removal from the lower troposphere. and others have indicated that there is a close relationship between methanotrophs and ammoniaoxidizing bacteria in soils. in part. the C and H isotope signatures of CH 4 are frequently adequate to reliably characterize bacterial or thermogenic natural gas types. based on Eq. Modified ‘Bernard’ diagram Žafter Bernard et al. but only more recently it has been accepted as an important sink of atmospheric methane Že. Ž1991. Univ. A clear understanding of these effects is necessary to relate the isotope signal of a natural gas to its source. through BMFT grants in Germany and by NSERC grant 105389 in Canada. Regensburg and R.. respectively. 1989. Carbon fractionation factors Ž ´ C . the C. in concert with the coexisting isotope information on CO 2 and H 2 O. In addition. such as mixing of different natural gases or where extreme substrate depletion and consumption occur. using chambers and incubations.. Mosier et al. Aalborg. Conrad. Ojima et al. Overall. Acknowledgements Collaboration with microbiologists.and H-isotopes of methane. could produce ambiguous methane isotope signals. found ´ C – CO 2 . ¨ Univ.. and Steudler and Whiticar Ž1998.. N. and Seiler et al. Max Planck Inst. using in situ gas microsampling of soil gases at Harvard Forest found carbon isotope effects for the consumption to be significantly lower than 5‰.

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