Carbon and hydrogen isotope systematics of …sethnewsome.org/sethnewsome/EE_files/Whiticar 1999.pdfCarbon and hydrogen isotope systematics of bacterial formation and oxidation of

Ž .Chemical Geology 161 1999 291–314www.elsevier.comrlocaterchemgeo

Carbon and hydrogen isotope systematics of bacterial formationand 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 moreeffectively by discerning changes in the dissolved constituents of the interstitial fluids, rather than by monitoring changes inthe bulk or solid organic components. The major dissolved carbon species in diagenetic settings are represented by the twocarbon redox end-members CH and CO . Bacterial uptake by methanogens of either CO or ‘‘preformed’’ reduced carbon4 2 2

Ž13 12 .substrates such as acetate, methanol or methylated amines can be tracked with the aid of carbon Cr C and hydrogenŽ 2 1 . Ž .DrH' Hr H isotopes. The bacterial reduction of CO to CH is associated with a kinetic isotope effect KIE for2 4

13 Ž .carbon which discriminates against C. This leads to carbon isotope separation between CO and CH ´ exceeding 952 4 C

and gives rise to d13C values as negative as y110‰ vs. PDB. The carbon KIE associated with fermentation ofCH4

Ž 13 .methylated substrates is lower ´ is ca. 40 to 60, with d C values of y50‰ to y60‰ . Hydrogen isotope effectsC CH4

during methanogenesis of methylated substrates can lead to deuterium depletions as large as d D sy531‰ vs. SMOW,CH4

Žwhereas, bacterial DrH discrimination for the CO -reduction pathway is significantly less d D ca. y170‰ to2 CH4

.y250‰ . These field observations have been confirmed by culture experiments with labeled isotopes, although hydrogenisotope exchange and other factors may influence the hydrogen distributions. Bacterial consumption of CH , both aerobic4

and anaerobic, is also associated with KIEs for C and H isotopes that enrich the residual CH in the heavier isotopes.4

Carbon fractionation factors related to CH oxidation are generally less than ´ s10, although values )20 are known. The4 CŽ .KIE for hydrogen ´ during aerobic and anaerobic CH oxidation is between 95 and 285. The differences in C and HH 4

isotope ratios of CH , in combination with the isotope ratios of the coexisting H O and CO pairs, differentiate the various4 2 2

bacterial CH generation and consumption pathways, and elucidate the cycling of labile sedimentary carbon. q 19994

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 ofmethanogenesis and methanotrophy continues to at-

) Fax: q1-250-721-6200; E-mail: [email protected]

tract the interest of microbiologists and geochemists,despite the fact that CH , even at saturated concen-4

trations in shallow organic-rich sediments, representsonly about 1‰ of the total organic carbon presentŽe.g., 10 m water depth, ca. 4% C , porosity ca.org

.0.9 . Apart from the more fundamental interests,

0009-2541r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0009-2541 99 00092-3

( )M.J. WhiticarrChemical Geology 161 1999 291–314292

such as bacterial physiology or organic matterremineralization, some of this curiosity reflects theeconomic fact that roughly 20% of the worldwidenatural gas reservoirs are from bacterial sourcesŽRice, 1992; Rice and Claypool, 1981; Whiticar,

.1994 . The bacterial CH cycle also receives current4

attention owing to its impact on the atmosphericŽ .CH budget Khalil and Shearer, 1993 . This follows4

Ž .the identification that CH in the atmosphere is: 14

a significant radiatively active or ‘‘greenhouse’’ gas,Ž .2 represents an important sink for tropospheric

Ž .hydroxyl radicals, 3 increasing in troposphericy1 Žconcentration at a rate of ca. 18 to 9 ppb yr e.g.,

. Ž .Dlugokencky et al., 1995 , and that 4 bacterial CH 4Ž .is the primary source ca. 80%–90% of this

Žatmospheric CH e.g., Cicerone and Oremland,4.1988; Whiticar, 1993 .

Furthermore, the processes of methanogenesis andaerobic CH oxidation are also attractive to study by4

microbiologists because they frequently involve onlysingle or two-carbon reactions. Thus, these bacterialpathways are more easily followed and reproducedin the laboratory. For the geochemist, methanogene-sis and methanotrophy are interesting because theyare sensitive indicators of the state of diagenesis in aparticular environmental setting.

The interest expressed in this paper reflects theintricate diagenetic relationships in the carbon cyclethat permit the most oxidized form of carbon toreside and be maintained adjacent to its most re-

Ž . Ž .duced form Fig. 1 . The physical depth separationof CH formation and consumption is often a transi-4

tional feature rather than distinctly zoned. Theactivity maxima of the respective CH formation4

and consumption processes can be separated spa-tially 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

Ženvironments e.g., Kuhl and Barker Jørgensen,.1992 .

An elegant method to track the complimentaryprocesses of methanogenesis and methanotrophy isprovided by the stable isotopes of C and H. Thepartitioning of the light and heavy isotopes of hydro-

Ž .gen and carbon e.g., Fig. 1 and the resultant isotopesignatures can be diagnostic for the identification ofmethane type and pathway. This paper reviews thestable isotope systematics primarily associated with

Fig. 1. Carbon cycle for bacterial methane formation and con-sumption. Numbers indicate the carbon and hydrogen isotope

Ž . Žfractionation factors ´ , ´ for the various mechanisms seeC HŽ ..Eq. 7 . MeOHsmethanol, Acsacetate, TMA s trimethyl-

amine and DMSsdimethylsulphide, DESsdiethylsulphide, ETsethanethiol.

the incorporation and release of C and H duringmethanogenesis and methylotrophy, i.e., the bacterialformation and consumption of CH .4

2. Methanogenic pathways

The microbiology, ecology and biochemistry ofthe various bacterial CH formation pathways have4

been reviewed extensively in several monographsŽe.g., Oremland and Capone, 1988; Zehnder, 1988;

.Garcia, 1990; Konig, 1992; Marty, 1992 .¨Methanogens, fermentative archaebacteria, are obli-gate anaerobes that metabolize only in anoxic condi-tions at redox levels Eh-y200 mV. The fact that

( )M.J. WhiticarrChemical Geology 161 1999 291–314 293

they cannot tolerate significant pO , nitrate or nitrite2

levels are due primarily to the instability of theirŽF –hydrogenase enzyme complex Schonheit et al.,¨420

.1981 . Methanogens form methane by pathwaysthat are commonly classified with respect to the typeof carbon precursor utilized by them. The primary

Ž .methanogenic pathways are referred to as: 1Ž .hydrogenotrophic – 52 species described, 2Ž .acetotrophic – 19 species described and 3

Žmethylotrophic – 10 species described e.g., Conrad.et al., 1985 . Hydrogeno-methylotrophic and

alcoholotrophic methanogens have also been dis-Ž .cussed Neue and Roger, 1993 . It is important to

remember that the anaerobic remineralization of or-ganic matter, ultimately to methane, relies on consor-tia of bacteria and microbes that successively break

Ž .down larger molecules. Conrad 1989 recognizedthat four bacterial assemblages operate in this com-

Ž .plementary fashion: 1 hydrolytic and fermentingŽ . q Ž .bacteria, 2 H -reducing bacteria, 3 homoaceto-

Ž .genic bacteria and finally 4 methanogenic bacteria.An approach, alternative to biochemistry, to clas-

sify methanogenic pathways uses microbial ecologi-cal relationships, namely methanogenic substrates.Methanogens utilize relatively few and simple com-

Ž .pounds to obtain energy and cell carbon: 1Ž .competitive and 2 non-competitive. The former are

those substrates which can be utilized more effec-tively by other bacterial assemblages, such as sul-

Ž .phate reducing bacteria SRB , and thus are eitherŽ .made unavailable out-competed or severely re-

stricted to the methanogens. Non-competitive sub-strates are those which are more suitable tomethanogens and less attractive to other bacteria,e.g., SRBs. Other factors, including redox potential,the availability of nutrients, substrates and terminalelectron acceptors, or consumption reactions can de-termine the occurrence of bacterial methane in aparticular environment. These competitive and non-competitive methanogenic pathways are outlined be-low.

2.1. CompetitiÕe substrates

ŽCompetitive substrates include CO reduced2.by hydrogen , acetate and formate, i.e., hydrogeno-

trophic, acetotrophic and methylotrophic pathways.

The first one, termed the ‘‘carbonate reduction’’pathway, can be represented by the general reaction:

CO q8Hqq8ey™CH q2H O, 1Ž .2 4 2

and the net reaction for the acetate fermentationpathway is:

UCH COOH™U CH qCO , 2Ž .3 4 2

where the U indicates the intact transfer of themethyl position to CH .4

Methanogenesis by competitive substrates isseverely restricted in water columns or sedimentpore fluids with abundant dissolved sulphate; typi-cally those systems with dissolved sulphate greaterthan 200 mM. In these sulphate-rich environments,SRB successfully outcompete methanogens for car-bon substrates, or in the case of carbonate reduction,for the available hydrogen.

In sulphate-rich zones, such as marine sedimentswhere methanogenesis is severely curtailed, methaneis generally present at only trace concentration levelsŽ .-0.5 mM . Most of the available labile carboncompounds in these zones are metabolized by non-methanogens and released, in part, to the dissolved

Ž .bicarbonate pool Fig. 2 . In addition, anaerobic CH 4

consumption in the sulphate zone also assists inmaintaining the low CH levels observed. Methan-4

otrophy is discussed in detail later.Once the available dissolved sulphate is ex-

hausted, usually at greater sediment depth, and theSRB become inactive, methanogenesis commencesusing competitive substrates. Carbonate reduction isthe dominant methanogenic pathway under theseconditions because of the relatively depleted levelsof other methanogenic substrates, such as acetate, bythe SRB and because of the accumulation of asizable dissolved bicarbonate pool in this sulphate-

Ž .free zone Fig. 2 . A generalized CH distribution4

for marine sediments with elevated organic carboncontent, i.e., shallow waters with high depositionalrates, is presented in Fig. 3.

Ž .In freshwater low sulphate environments,methanogenesis proceeds uninhibited once anaerobic

Ž .conditions are established Fig. 3 . Competition bythe SRB is essentially absent so that the short-chained

Ž .volatile fatty acid VFA pool is available and pro-vides, in addition to acetate or methylated amines,


Fig. 2. Schematic diagram showing methanogenic pathways in marine and terrestrial environments.

Žimportant substrates for methanogenesis Fig. 2, e.g.,.Marty, 1992 . Methanogenesis by carbonate reduc-

tion in freshwater environments is initially less sig-nificant, but increases in importance as the othersubstrate pools become exhausted, and the

Ž .methanogens excepting the obligate methylotrophsswitch over to or start reducing the bicarbonatecarbon source with hydrogen. Previous investigatorsstated that acetate fermentation is responsible forroughly 70% of the methanogenesis in freshwater

Ž . Ž .environments, e.g., Koyama 1964 , Takai 1970 ,Ž . Ž .Belyaev et al. 1975 , Winfrey et al. 1977 and

Ž .Cappenberg and Jongejan 1978 . The utilization ofother methylated substrates andror carbonate reduc-tion are suggested as constituting the remainder.Similar results have been cited for sewage sludges

Žand from culture studies Bryant, 1979; Zehnder et.al., 1982 .

2.2. Non-competitiÕe substrates

Non-competitive substrates for methanogenesisinclude both the acetotrophic and methylotrophicpathways that utilize the carbon substrates methanol,methylated amines, such as mono-, di- and tri-meth-

Ž .ylamines MA, DMA and TMA, respectively , andcertain organic sulphur compounds, e.g., dimethyl-

Žsulphide DMS; Daniels et al., 1984; Kiene et al.,.1986 . A typical fermentation reaction for these sub-

strates by methylotrophic methanogens can generallybe represented by:

CH yAqH O™CH qCO qAyH, 3Ž .3 2 4 2


Fig. 3. Sediment depth profiles of methane concentration and carbon isotope composition in marine and freshwater environments. Asoxiczone, Bssulphate reduction zone, Csmain methanogenic zone, Dszone of substrate depletion andror shift to alternative substrate formethanogenesis, e.g., carbonate reduction.

The relative importance of some of these meth-ylated substrates as the source for bacterial CH is4

currently uncertain. However, CH formation utiliz-4

ing non-competitive substrates is known for environ-ments such as rice paddies, salt marshes and otherwetlands, i.e., those environments which are knownto be major emission sources of CH to the atmo-4

Žsphere Cicerone and Oremland, 1988; Andreae and.Schimel, 1989; Whiticar, 1990 . Enhanced methano-

genesis has also been obtained in culture experi-ments by additions of various ethylated and sulphide

Ž .compounds Oremland et al., 1988b includingŽ . Ž .diethylsulphide DES , methanethiol MeSH ,

Ž . Ž .ethanethiol EtSH and ethanol EtOH . Their impact

as substrates for methanogens is also currently un-known, but they are thought to be less important.

3. C and H isotope variations of methane innatural environments

The isotope data are reported in the standardŽ 13 .d-notation e.g., d C, d D expressed here in permil

Ž .‰ :

RŽ . samplea 3d s y1 10 ‰ , 4Ž . Ž .x RŽ .a standard


13 12 Ž 2 1 .where R is the Cr C or DrH ' Hr H ratiosa

relative to the PDB and SMOW standards, respec-tively.

The study of C and H isotope systematics basedon in vivo observations of methanogenesis have, inthe past, generally emphasized the competitive sub-

Ž .strates. Whiticar et al. 1986 used the combinationof C and H isotope data of CH , together with those4

of the coexisting bicarbonate and water species, todefine compositional fields for the differentmethanogenic pathways. Using field measurementsof CH from various recent and ancient sedimentary4

environments, including marine, marsh, swamp andlacustrine settings, it was also possible with stable Cand H isotopes to differentiate between the differentbacterial and thermogenic CH types. The combina-4

tion of d13C and d D values defining theCH CH4 4

various natural sources of CH in the geosphere is4

Ž .demonstrated in the CD-diagram Fig. 4 . The atmo-spheric methane CD pair does not fall within any ofthe fields in Fig. 4 due to the isotopic offset causedby the C and H isotope effects associated with thephotochemically mediated OH-abstraction reactionŽ .e.g., Whiticar, 1993 .

3.1. Thermogenic, non-bacterial, methane

Thermogenic CH is generally, but not exclu-4

sively, enriched in 13C compared with bacterial CH .4

The former has a d13C range extending fromCH 4

Ž .roughly y50‰ to y20‰ Fig. 4 . The dissimilarityin isotope distributions of bacterial and thermogenicCH accumulations is related to several factors in-4

cluding precursor compounds, the difference in typeŽ .and magnitude of kinetic isotope effects KIE in-

volved, and to the generally higher temperatures for

Fig. 4. CD-diagram for classification of bacterial and thermogenic natural gas by the combination of d13C and d D information.CH CH4 4


the thermogenic generation of hydrocarbons. Withinthe range of d

13C values known for typical ther-CH 4

mogenic gases, it is possible to further classify themŽ .according to the source rock type kerogen type and

maturity level from which they were derived. Adetailed discussion of isotope variations for thermo-

Žgenic gases exceeds the framework of this paper see.Schoell, 1988; Whiticar, 1994 . However, in sum-

mary the d13C of thermogenic gases becomesCH 4

progressively enriched in 13C with increasing matu-rity, eventually approaching the 13Cr12 C of the orig-

Žinal organic matter or kerogen and rarely even.heavier . In general, the carbon isotope separation

between thermogenic CH and organic matter4Ž 13 13 .d C –d C can vary from ca. 30‰ to 0‰.CH C4 org

Furthermore, hydrogen-rich organic matter, i.e.,kerogen types I and II, can often generate CH at4

lower levels of thermal stress and with more nega-13 Žtive d C values than, for example, coaly typeCH 4

.III kerogen sources. The hydrogen isotope ratios ofthermogenic CH range from d D values of ap-4 CH 4

Ž .proximately y275‰ to y100‰ Fig. 4 . Due to theconsiderable overlap in d D between certain bac-CH 4

terial and thermogenic methane types, the hydrogenisotope ratios of CH are less useful as an isolated4

parameter to classify natural gases. However, if theyare used in combination with additional molecular orisotope compositional gas data, e.g., C –C , the2 4

d D information can be particularly valuable. InCH 4

addition to thermogenic methane, the C- and H-iso-tope signatures and ranges of other non-bacterialmethanes, such as geothermal and hydrothermal, arepresented in Fig. 4 for comparison.

3.2. Bacterial methane

The term ‘bacterial’ is preferred over ‘biogenic’because the carbon in both bacterial or thermogenicnatural gases, including most methane, is ultimatelyderived from or has been part of the biological loopof the exogenic carbon cycle.

Bacterial CH has a wide range of C and H4Ž . 13isotope ratios Fig. 4 , varying in d C fromCH 4

y110‰ to y50‰, and in d D from y400‰ toCH 4

y150‰. The bacterial fields in the CD-diagramshown in Fig. 4 are delineated by compiled CH 4

Ždata from natural environments Whiticar et al.,.1986 . Within this large range of values two primary

bacterial CH fields were initially identified, namely,4Ž . Ž .methane that is formed in 1 freshwater and 2

saline sedimentary environments. These fields arenow labeled in Fig. 4 as bacterial methyl-type fer-mentation and bacterial carbonate reduction, respec-tively. Bacterial CH accumulated in marinersaline4

environments is generally more depleted in 13C andenriched in deuterium than observed in freshwater

Ž .environments Fig. 4 .This initial distinction was based only on the

classification of the depositional environments anddid not explicitly consider the methanogenic path-

Ž .way s involved. However, methanogenic path-ways can also be inferred from the C and H isotopedata. This relies on the combination of environ-mental information with the understanding ofmicrobiological processes presented in Section 2,i.e., that carbonate reduction is the dominantmethanogenic pathway in marine environments and

Ž .methyl-type substrates acetate are more importantin freshwater environments.

The separation of the two bacterial CH fields in4

the CD-diagram can be made at approximate bound-aries of d

13C of y60‰ and d D of y250‰CH CH4 4

Ž .Fig. 4 . Although it is common to measure only thecarbon isotopes of CH , the hydrogen isotope ratios4

of CH are particularly helpful in defining the bacte-4

rial CH types. In certain instances, the CH isotope4 4

data have a ‘‘transitional’’ isotope composition thatlies between the two fields. The explanation for this

Ž .overlap is related to the combined effects of: 1Ž .kinetic isotope fractionation by methanogens, 2

mixtures of various pathways andror CH types4Ž . Ž .e.g., migrationrdiffusion and 3 variations in C-and H-isotope composition of precursor organic mat-ter. These three explanations are expanded in Section4.

4. Factors controlling isotope signatures inbacterial methane

4.1. Kinetic isotope effects for carbon

The distribution of carbon isotopes during uptakeand metabolism of carbon compounds by metha-nogens is controlled primarily by 1. isotope signa-ture of source material and 2. KIEs. In the general


Table 1Magnitude of carbon isotope effects associated with methanogenesis

Ž .A Methanogenesis: natural observations

Ž .Environment Fractionation factor ´ of carbon dioxide–methaneC

Ž .Freshwater methylated compounds 39 to 58Ž .Marine carbonate reduction 49 to 95

Ž .B Methanogenesis: culture experiments

Ž .Substrate Fractionation factors Ć

Substrate–methane Carbon dioxide–methane

Methanol 68 to 77 40 to 54Acetate 24 to 27 10TMA 39 49CO –H 55 to 58 55 to 582 2

DMS 44 to 54DES 49EtSH 54

case for each particular compound, the moleculesŽ 12with the lower isotopic mass e.g., CH COOH as3

13 .opposed to CH COOH diffuse and react more3

rapidly and thus are utilized more frequently than theisotopically heavier species. This discrimination ac-counts for the strong depletion in 13C of bacterialCH relative to the precursor substrates. The appar-4

ent magnitude of this isotope fractionation varies forŽ .different pathways Table 1 . For example, the fer-

mentation of acetate involves an enrichment in 12 Con the order of 25‰ to 35‰ for d

13C relative toCH 4

d13C . A significantly greater 12 C enrichmentacetateŽ 13 13 .over 55‰ for d C relative to d CCH carbon dioxide4

is observed for methanogenesis by carbonate reduc-tion.

In a closed system, the continued preferentialremoval of the isotopically lighter molecules fromthe carbon pool during methanogenesis results in aprogressive shift in the residual substrate towardsheavier, 13C-enriched values. Hence, there is a corre-sponding progressive 13C enrichment in CH that is4

subsequently formed. This carbon isotope mass bal-ance is commonly described by the distillation func-

Ž .tions of Rayleigh 1896 , that can be approximatedŽ Ž . Ž ..by the general forms Eqs. 5 and 6 to describe

the isotope ratio of the remaining reactant and accu-mulated product, respectively:

R sR f Žay1. 5Ž .r , t r , i

and

1y f aŽ .R sR 6Ž .p , t r , i 1y fŽ .

where R is the isotope ratio of the precursorr, x

substrate, e.g., acetate or CO , and accumulating2Ž .initially i and at time t, and R . is the isotopep, x

ratio of the accumulating product, e.g., methane, f isthe fraction of initial substrate remaining at time tand a is the kinetic isotope fractionation factor.

The isotope separation in d-notation between twocompounds, e.g., d

13C and d13C , can be ex-CO CH2 4

Ž .pressed as the isotope separation factor ´ :C

´ f103 ln a f103 a y1Ž .C C C

fd13C yd

13C . 7Ž .CO CH2 4

Ž .Note that the approximations in Eq. 7 are validfor relatively small isotope separations, typically lessthan 25‰ between species. For larger separations,common with hydrogen isotopes, these assumptionscan lead to significant errors. In these cases, thefractionation factor a is better defined as the ratioC

of the reaction rate constants for the two isotopes,Ž . 13 12e.g., referring to Eq. 1 , a s kr k, where:C

12k 13k12 12 13 13CO ™ CH and CO ™ CH . 8Ž .2 4 2 4


For equilibrium isotope situations, the relationshipbetween a and d is defined as:

d q103Ž .Aa s . 9Ž .Ay B 3d q10Ž .B

Normally, isotope equilibrium cannot be assumedfor these kinetic reactions. However, this notationhas sometimes been used to express the magnitudeof the isotope partitioning. These Rayleigh

Ž Ž . Ž ..distillation functions expressions Eqs. 5 and 6can be converted to d-notation and simplified by the

Ž Ž ..substitution with ´ Eq. 7 and taking Fs1y f ,the remaining reactant, where:

d13C sd

13C q´ ln 1yF , 10Ž . Ž .substrate , t substrate , i

and for the instantaneous product formed:

d13C sd

13C q´ 1q ln 1yF .Ž .Ž .methane , t substrate , i

11Ž .Ž .The actual location s , e.g., at the membrane

andror enzymatic level, where the isotope fractiona-tion occurs during methanogenesis is still uncertain.However, the consequence of this fractionation isthat within a specific environment a considerable

Fig. 5. Theoretical shifts in d13C and d D as a result of isotope variations in original organic matter and through secondary effectsCH CH4 4

Ž .substrate depletion and methane oxidation .


range in carbon isotope values can be observedwhich is dependent on the degree to which the

Ž .substrate has been depleted Fig. 5 . In cases ofextensive substrate exhaustion, the carbon isotopevalues of bacterial CH can approach those of the4

original organic matter. In some instances wheremass balance is not maintained, such as loss of themethane initially formed, the methane can in fact bemore enriched in 13C than the starting precursormaterial. Although such variations in the d

13CCH 4

values exist, they are most commonly observed inmethanogenic culture experiments.

4.2. Temperature effects on carbon isotope Õalues

The effect of temperature on the fractionation ofcarbon isotopes associated with methanogenesis is

Ž .poorly understood. Conrad and Schutz 1988¨demonstrated a 2.5- to 3.5-fold increase in CH 4

generation due to a 108C temperature rise, but it isnot known if this would affect the KIE. Later, theyreported that the temperature could influence the

Žrates and pathways of methanogenesis Schutz and¨.Conrad, 1996; Schutz et al., 1997 . Whiticar et al.¨

Ž .1986 showed that there is a general trend to lowerfractionations with increasing temperature. Empiri-cally, one could expect a decrease in the isotopeeffect at higher temperatures. A rough, albeit incom-plete, test is provided by comparing the a betweenC

CO and CH from carbonate reducing methanogen-2 4Ž .esis at various temperatures Table 2 . This relation-

ship, shown in Fig. 6, indicates, as expected fromthermodynamic considerations, that the KIE de-creases significantly with increasing temperature.Over a 1108C temperature rise, the isotopic partitionof d

13C –d13C decreases from 100‰ to 40‰,CO CH2 4

equivalent to an ´ change of ca. 86 to 39 forCŽ Ž .. Ž .CO –CH pair Eq. 7 . Botz et al. 1996 also2 4

documented a temperature dependence of isotope

fractionation during biological methanogenesis. Theirculture experiments of methanogenesis by carbonatereduction with Methanococcales at 35–858C gave´ values of 76 to 47, respectively.C

In addition to temperature, it has been suggestedthat the carbon isotope fractionation is dependent onother factors such as substrate concentration and

Ž .rates of methanogenesis. Zyakun 1992 reported forculture experiments that the magnitude of carbonisotope fractionation during methanogenesis by car-bonate reduction was dependent on the CO gassing2

rate. Above a critical CO threshold the CO –CH2 2 4

isotope separation was constant, but decreased dra-matically as the amount of CO available dropped.2

Ž .Fuchs et al. 1979 demonstrated a similar result. InŽ .addition, Zyakun 1992 stated that the magnitude of

the carbon isotope fractionation is also dependent onthe rate of methanogenesis by a variety of

Ž .methanogens and substrates CO and methylated .2

It is clear from these experiments that the isotopeseparation between the precursor and methane de-creases with the degree of substrate utilization. How-ever, the magnitude of separation is unlikely to becaused by a change in the isotope effect, i.e., achange in the enzymatic kinetic reaction rates for 13C

12 Ž Ž ..vs. C Eq. 8 . It is more likely that the decreasein precursor-methane isotope separation is due to a‘reservoir effect’ caused by substrate depletionandror diminished rates of substrate transport acrossthe membrane walls of the methanogens. Thus, themaximum expression of the carbon isotope effectcorresponds to the situations with high substratelevels. Increased levels of methanogenesis at highertemperatures is well documented. For example,

Ž . Ž .Martens et al. 1986 and Jedrysek 1995 showedthat diurnal variations in temperature could influencethe carbon isotopes, but again this is probably amethanogenic rate effect and not enzymatic. Iversen

Table 2Effect of temperature on the magnitude of carbon isotope effects associated with methanogenesis by carbonate reduction

Ž .Locationrbacterium Temperature 8C ´ CO –CH ReferencesC 2 4

Ž . Ž .Bransfield Strait Antarctic y1.3 86 to 95 Whiticar and Suess 1990Ž .Methanosarcina barkeri 20 77 Whiticar, Muller, Blaut unpublished data¨

37 58Ž .Thermoautotrophicum 65 34 to 40 Fuchs et al. 1979

Ž .Hyperthermophil 110 40 Whiticar, Stetter, Huber unpublished data


ŽFig. 6. Temperature dependence of carbon isotope partitioning Ć13 13 .between d C and d C by methanogens via the carbon-CO CH2 4

ate reduction pathway.

Ž .1996 found that the temperature may have someeffect on the rate of methane oxidation but theresults were inconclusive. There is no evidence indi-cating that temperature affects the carbon or hydro-gen isotopes related to methane oxidation, althoughsome effect, albeit small, could be anticipated.

Temperature was suggested by Hornibrook et al.Ž .1997 to influence the methanogenic pathway andthe carbon and hydrogen isotope signatures. Waldron

Ž .et al. 1998 challenged this and in reply HornibrookŽ .et al. 1998 subsequently clarified that temperature

indirectly exerted some influence on substrate avail-ability due to enhanced microbial remineralizationrates.

4.3. Hydrogen isotope effects

Our understanding of the hydrogen isotope effectsassociated with methanogenesis is limited. Empiricalevidence suggests that substantial enrichments in 1Hdo occur as hydrogen is transferred to the methanemolecule from either organic precursors or formationwater. This hydrogen isotope partitioning is presentregardless of the methanogenic pathway, but the

Žmagnitude may be dependent on it, Whiticar et al.,.1986; Burke et al., 1988a . As expected by the larger

mass difference between 1H and 2 H than 12 C vs.13C, the hydrogen isotope partitioning is larger thancarbon isotopes. As discussed below, it remains un-

resolved to what extent kinetic vs. equilibrium mech-anisms dominate the hydrogen isotope distributionsin methane. In addition, there are suggestions that

Žfactors such as hydrogen concentration Burke, 1992,. Ž1993 or hydrogen isotope exchange de Graaf et al.,.1996 complicate the situation.

4.4. Mixtures

Bacterial CH with ‘‘mixed’’ or ‘‘transitional’’4

isotope signatures can often be present in a gassample. This could be the result of contributions of

Ž .CH from different methanogenic pathways Fig. 54

which may be operating in the same location, eitherconcurrently or successively. For example, the formof methanogenesis can shift from one substrate toanother, such as the succession to carbonate reduc-tion after the acetate pool is exhausted. This is

Žobserved in some freshwater systems e.g., Horni-.brook et al., 1997 . Work in shallow marine sedi-

Ž .ments by Sansone and Martens 1981 , Martens et al.Ž . Ž . Ž .1986 , Burke et al. 1988b and Kelley et al. 1992demonstrated that shifts to different methanogenicsubstrates or isotope signatures might also be con-trolled by seasonal conditions.

These gas mixtures could also be the result ofphysical mixing of different CH pools in response4

to vertical or lateral migration or diffusion of gas.This mixing is not necessarily restricted to bacterialgas, but can also involve admixtures of thermogenicgas.

4.5. Isotope Õariations in precursor organic matter

It is more difficult to assess the third point,namely, variations in isotope composition of theprecursor organic matter. C and H isotope values ofbulk organic matter that are available for specificenvironments exhibit the approximate isotope rangesshown in Fig. 5. With the exception of dissolved

Ž .ÝCO see below , isotope measurements are sparse2

for specific methanogenic substrates in natural envi-ronments. The magnitude to which the C and Hisotope compositions of the various methanogenicsubstrates deviate from those of the bulk organicmatter is not well known, nor is the magnitude ofany intramolecular isotope fractionation present in


Žsuch substrates well understood Yankwich andPromislov, 1953; Meinschein et al., 1974; Rossmann

.et al., 1991 . However, it has been assumed up tonow that the natural C and H isotope signatures of asubstrate in a specific setting are similar to those ofthe bulk organic matter, provided that severe sub-strate depletion has not occurred and that large inter-or intramolecular variations are not present. Analyti-cal techniques now are available to measure C and H

isotope ratios in methane and the precursors in theŽ .picomole range e.g., Whiticar et al., 1998a,b , that

should help resolve this issue.Methylated substrates available for fermentation

by methanogens often are present only in minoramounts. This is due to an effective combination offormation and utilization that leads to a relativelyshort residence time. Although this may control therate of methanogenesis via methyl-type fermentation,

Ž . Ž .Fig. 7. a Concentration depth profiles of methane, bicarbonate and sulphate and b carbon isotopes of methane and bicarbonate in themarine sediments from core 1327, Bransfield Strait, Antarctic.


the amount of methane produced can exceed satura-Ž .tion Fig. 3 . The low concentration of methanogenic

substrates, such as organic acids, makes it difficult toŽisotopically model their removal e.g., Sansone and

.Martens, 1981; Blair and Carter, 1992 . In contrast,organic-rich marine sediments can have substantial

Ž .bicarbonate concentrations ) 100 mM . Theseamounts permit more readily the modeling of carbonisotope systematics for methanogenesis by carbonate

Ž .reduction. Claypool 1974 provided one of the firstmodels using gas and interstitial data from sediment

Žcores of the Deep Sea Drilling Program DSDP, later.Ocean Drilling Program, ODP . An illustration of

results similar to those found in numerous DSDP,ODP and other sediment cores is illustrated by the7.5-m piston core taken at station 1327 in the Brans-

Ž .field Strait, Antarctic Whiticar and Suess, 1990 . Inthis case, CH concentrations first exceed 100 ppb4Ž .weight of gasrweight of wet sediment beneath the

Ž .sulphate reduction zone Fig. 7a . The continualremoval of dissolved bicarbonate by methanogens ismarked by the reduction in the increase of dissolvedÝCO concentration with depth from the sulphate2

reduction zone to the zone of methanogenesis. Thischange was accompanied by a sharp decrease in

Ž .molar C:N ratio carbonate alkalinity:ammonia dueto the preferential uptake of carbon over nitrogen.The more rapid uptake and utilization of 12 C bicar-bonate by methanogens in core 1327 leads to agradual shift in d

13C towards heavier values inCO 2

Ž .the older sediments Fig. 7b . This depletion is inturn expressed in the d

13C of the CH , which also4

becomes heavier with sediment depth in themethanogenic zone as predicted by the Rayleigh

Ž Ž ..function Eq. 15 . The carbon isotope separationbetween CO and CH in the CH zone of core2 4 4

1327 is consistently around 80‰. The fact that theCO concentration does not substantially decrease in2

this zone, indicates that CO is continually added by2

organic remineralization to the pool in themethanogenic zone, although overall there is a 12 Cdepletion by the methanogens. This strict relation-ship between d

13C and d13C provides impor-CO CH2 4

tant information about methanogenic pathways, as isdeveloped in greater detail below.

The variations in inter- and intramolecular isotopedistributions and effects for hydrogen in organicmatter are much less well constrained than for car-

bon. Typically only determinations of bulk d areD Corgavailable for natural environments without the re-spective DrH values for specific substrates or hy-drogen isotope distributions within the precursormolecules. Some culture experiments have used ad-ditions of water and substrates with known hydrogenisotope values to assess the methanogenic processesand magnitudes of the related isotope effects.

5. Isotope systematics of methanogenesis with thecoexisting CO and water2

5.1. Carbon isotopes

Ž .Although the CD-diagram Fig. 4 can be used todifferentiate the major CH sources, the various4

secondary effects discussed above can obscure theŽ .signature of source s under certain conditions. The

ambiguity in determining the methanogenic pathwaycan sometimes be resolved by considering the iso-

Ž 13 .tope information of CO d C and formation2 CO 2

Ž .water d D which coexist with the CH , e.g., inH O 42

the corresponding pore fluid or water mass.In the case of carbon, the isotope separation factor

13 13 Ž .between d C and d C ´ is defined byCO CH C2 4

Ž .Eq. 7 . In natural marine or saline environments Ć

associated with methanogenesis predominantly bycarbonate reduction ranges from 49 to over 100, with

Žvalues most commonly around 65 to 75 Fig. 8, after.Whiticar et al., 1986 . In comparison, the corre-

sponding fractionation factors for methanogenesis infreshwater environments, i.e., those dominated byfermentation of methylated substrates, are distinc-tively lower with ´ values typically ranging be-C

Ž .tween 40 and 55 Fig. 8 . A key point is that thiscarbon isotope fractionation factor tends to remainmore or less constant for each specific setting ordiagenetic environment. Thus, although the d

13CCH 4

can shift dramatically in response to the changingisotope ratio of the depleting substrate, the isotopeseparation between CH and CO remains consistent4 2

for, and indicative of, the particular methanogenicpathway. This is illustrated in Fig. 8 by the produc-tion arrow. Similarly, the environments wheremethanogenesis operates with mixed substrates canalso be recognized using the CH –CO pairs despite4 2

large variations in the actual d13C values.CH 4


13 13 Ž . Ž .Fig. 8. Combination plot of d C and d C with isotope fractionation lines ´ according to Eq. 7 . Methanogenesis by carbonateCH CO C4 2Ž . Ž .reduction saline, marine region has a larger CO –CH isotope separation than by methyl-type fermentation freshwater region or methane2 4

consumption. The figure also shows the observed CO –CH carbon isotope partitioning trajectories resulting from both bacterial methane2 4

formation and oxidation processes.

The carbon isotope fractionation factors betweenCO and CH , associated with methanogenesis in2 4

marine and freshwater environments, are summa-rized in Table 1. For comparison, Table 1 alsoprovides the carbon fractionation factors between

ŽCH and various substrates after Oremland et al.,4.1988a,b; Whiticar, 1994 , as determined by culture

experiments. Similar to that reported by RosenfeldŽ . Ž .and Silvermann 1959 and Krzycki et al. 1987 ,

methanol exhibits the largest carbon isotope fraction-ation between a methylated substrate and the methane

Ž .formed, i.e., ´ s68 to 77 Table 1 . Pre-C methanol – methane

dictably, the largest CO –CH separation was ob-2 4Ž .served for CO -reduction, ´ s58 Table 1 . This is2 C

comparable to the carbon isotope fractionation valueŽ .of ´ ca. 57 reported by Zyakun 1992 for carbon-C

ate reduction culture with CO concentration as2

above and independent of the critical threshold. Sim-Ž .ilarly, Waldron et al. 1998 found ´ to be ca. 55C

for carbonate reduction in their batch cultures. In the

same paper, the authors reported ´ to be ca. 24 forC

acetate fermentation.

5.2. Hydrogen isotopes

The relationship between the hydrogen isotoperatios of bacterial CH and the coexisting formation4

water can also be employed to track methanogenicpathways. This consideration, introduced by Nakai et

Ž . Ž .al. 1974 and developed by Schoell 1980 , Wolte-Ž . Ž .mate et al. 1984 and Whiticar et al. 1986 , is due

to methanogens deriving a specific proportion oftheir hydrogen for CH formation ultimately from4

the water in which they live. For carbonate reduc-tion, the coexisting formation water is essentially the

Ž .initial hydrogen source 100% . This has been con-Ž .firmed experimentally by Pine and Barker 1956 ,

Ž . Ž .Fuchs et al. 1979 and Daniels et al. 1980 . Thefermentation of methylated substrates is thought toinvolve an intact transfer of the three methyl hydro-


Ž . Ž .gens 75% to the CH molecule Fig. 9 . This intact4

hydrogen transfer has been demonstrated in cultureŽstudies with deuterated substrates Pine and Barker,

1956; Pine and Vishniac, 1957; Daniels et al., 1980;.Whiticar et al., 1998a,b .

The empirical relationship expected betweend D and the coexisting d D as depicted in Fig.CH H O4 2

9 should be:

d D sm d D yb , 12Ž .Ž .CH H O4 2

where ms1 holds for carbonate reduction, and ms0.25 is expected for the fermentation of methylated

Ž .substrates. The values for ‘b ’ in Eq. 12 are depen-dent on the isotope effects involved in the abstrac-tion and transfer of the hydrogen. For the methylatedcompounds, they are also additionally dependent onthe hydrogen isotope ratio of the methyl hydrogens.The combined hydrogen isotope effects during car-bonate reduction in various environments appear tobe remarkably consistent around 160‰ to 180‰ for

Fig. 9. Sources of hydrogen incorporated into methane through thecarbonate reduction and methyl-type fermentation methanogenic

Ž .pathways. The letters refer to pathways: a is the direct utilizationof water–hydrogen during carbonate reduction, with an associated

Ž . Ž .isotope effect ´ of 160; b is the direct transfer of methyl–hy-HŽ . Ž .drogen from the organic matter d D ca. y80‰ ; c is theorg

dissociation of water to hydrogen gas either inorganically or viaŽ .biological processes; d is the direct synthesis of hydrogen gas by

Ž .bacterial activity, e.g., acetoclasts; e is the incorporation ofŽhydrogen gas as the final hydrogen into the methane molecule the

. Ž .magnitude of the isotope effect is poorly constrained ; f is apotential direct utilization of water by methanogens during fer-mentation.

CH relative to the formation water, as shown in4

Fig. 10.There is probably not a universal value for b with

respect to methylated substrates. Rather, the magni-tude of the isotope offset is site specific. However,most studies suggest that b associated withmethanogenesis by methyl-type fermentation is sig-nificantly larger than for carbonate reduction. Based

Žon the current SEOS School of Earth and Ocean. ŽSciences, Victoria and BGR Bundesanstalt fur Ge-¨

.owissenschaften und Rohstoffe, Hannover databases, values of b known for methylated substratesare greater than 300 and extend up to 377.

In methanogenic environments with more thanone active pathway, the situation is more compli-cated. To accommodate the mixture of two path-ways, e.g., combination of carbonate reduction andfermentation of methylated substrates, in the isotope

Ž .mass balance, Eq. 7 can be modified to:

d D s f d D y160Ž .CH H O4 2

q 1y f 0.75d DŽ . Ž .methyl

q 0.25d D , 13Ž .Ž .hydrogen

where f is the relative proportion of methanogenesisŽ .by carbonate reduction fs0 to 1 to fermentation

by methylated substrates, d D is the hydrogenmethyl

isotope ratios of the intact-transferred methyl groupand d D is that of the final hydrogen incorpo-hydrogen

rated. d D and d D are readily measured, butCH H O4 2

our present deficiencies in precise information on thedistribution of hydrogen isotopes in methylated sub-

Ž .stances make solution of Eq. 13 difficult. However,ranges on the parameters can be estimated.

In recent years, the hydrogen isotope systematicsassociated with methanogenesis have been revisited.

Ž .Sugimoto and Wada 1993, 1995 investigated therelationships between d D and d D for riceCH H O4 2

paddy soil during methanogenesis in culture experi-ments. Although they only indirectly calculated therelative importance of the two methanogenic path-ways, the authors reported values for m and b

Ž . Žaccording to Eq. 12 of 0.437 and 302 L4 in Fig..10 , respectively, for methyl-type fermentation. In-

terestingly, they also calculated m and b values of0.683 and 317 for the carbonate reduction pathwayŽ .L6 in Fig. 10 . These latter culture values contraststrongly with those known empirically for natural


Ž .Fig. 10. Dependence of hydrogen isotope ratio in methane d D as a function of the hydrogen isotope ratio of the coexisting formationCH 4Ž . Ž . Ž .water e.g., porewater, water column after Eqs. 12 and 13 . Methanogenesis by carbonate reduction follows a slope ‘‘m’’ of 1 with an

Ž .isotope offset ‘‘b ’’ of ca. 160‰ L1, F:Rs0:100 . The methyl-type fermentation pathway has a slope m of ca. 0.25 and an offset b ofŽ . Ž Ž .300‰ to 370‰ L2, F:Rs100:0 . L3 shows the typical d D –d D relationship observed by Whiticar et al. 1986 for freshwaterH O CH2 4

Ž . Ž . Ž . Ž .F:Rs80:20 . The relationships of Sugimoto and Wada 1995 for methyl-type fermentation L4 and carbonate reduction L6 and ofŽ . Ž . Ž .Waldron et al. 1998 for a mixture of the two pathways L5 are shown as dashed lines. Additional data of Balabane et al. 1987 , Burke et

Ž . Ž .al. 1988a , and Grossman et al. 1989 are shown as points 7–9.

environments. The explanation offered by SugimotoŽ .and Wada 1995 for the difference in the carbonate

reduction values between freshwater and marine en-vironments was that the former has higher hydrogenconcentration and longer hydrogen residence time. A

Ž .similar effect was proposed by Burke 1993 . Horni-Ž .brook et al. 1997 reported that their natural wetland

Ž .studies fit the model of Whiticar et al. 1986 . Theycountered that in comparison with natural settings,the high H concentrations in the culture experi-2

Ž .ments of Sugimoto and Wada 1995 may haveaffected the magnitude of the hydrogen isotope parti-tioning and led to the observed discrepancies. Burke

Ž . Ž .et al. 1988a and Grossman et al. 1989 foundd D –d D relationships indicating mixed reac-H O CH2 4

Ž .tions Fig. 10, points 7 and 8, respectively whereas

Ž .the culture study of Balabane et al. 1987 gaved D –d D values similar to methyl-type fer-H O CH2 4

Ž .mentation Fig. 10, point 9 .Ž .Sugimoto and Wada 1995 suggested that hydro-

gen isotope exchange could explain the difference inŽ Ž ..m and b values Eq. 12 for methyl fermentation

Ž .between their results 0.437 and 302 and those inŽ . Ž .Whiticar et al., 1986 0.25, 321 . Support for hy-drogen isotope exchange during acetate fermentation

Ž .comes from the work of de Graaf et al. 1996 .Certainly some of the methanes strongly depleted indeuterium, i.e., those lighter than d D ca.CH 4

y400‰, are difficult to explain by direct methylhydrogen incorporation. For example, if one assumesd D value of y120‰ for the three methyl hydro-org

gens donated, and a d D of y400‰, this wouldCH 4


Ž .give by Eq. 13 a non-sensical d D value ofHŽ .y1240‰ for the final hydrogen Fig. 11 . This

indicates that either the hydrogen in the precursormethyl group is more depleted in deuterium thanbulk organic matter or some isotopic exchange hasoccurred.

Ž .Waldron et al. 1988 suggested that the hydrogenisotope signature in bacterial methane is set by theenzymatic incorporation of hydrogen from water andnot by the precursor hydrogen on the methyl group.They proposed that this enzymatic control is opera-tive for both acetoclastic and carbonate reductionmethanogenic pathways. They calculated the magni-

Ž .tude of this isotope fraction a to be 1.47.H O – CH2 4

Ž . ŽWaldron et al. 1988 found values for m and b Eq.Ž ..12 of 0.623 and 319 for cultures of landfill mate-rial with both methanogenic pathways operative at

Ž .unknown relative proportions L5 in Fig. 10 .The processes controlling the distribution of hy-

drogen in methane during methanogenesis remainunclear. Certainly, numerous questions require de-

Fig. 11. Comparison of hydrogen isotope ratio in organic matterŽ .d D as a function of the hydrogen isotope ratio of the finalorg

Žhydrogen attached during methyl-type fermentation after Eqs.Ž . Ž ..12 and 13 . The lines are the mass balance for the variousvalues of the resultant d D .CH 4

tailed attention, including that of possible hydrogenisotope exchange, effects of hydrogen partial pres-sures, rates of methanogenesis, and the translation ofresults from culture experiments to natural environ-ments.

6. Isotope effects associated with bacterialmethane oxidation

Bacterial consumption is a major sink of CH in4

the geosphere and in water columns. Without thishydrocarbon metabolism, the flux of CH into the4

atmosphere could be orders of magnitude greater,and its impact on the global atmospheric thermal andchemical budgets would be even more significantŽ .Whiticar, 1993 . Bacterial CH consumption can be4

by either aerobic or anaerobic oxidation. The formeris a common process in freshwater settings normallyat the anoxicroxic interface, for example withinsoils or within the uppermost centimeters of sedi-

Žments in swamps or lakes e.g., Rudd and Hamilton,1975; Bossard, 1981; Conrad, 1989; Steudler et al.,

.1989 . Anaerobic CH consumption was controver-4

sial for a long period amongst microbial ecologists,but the arguments presented for anaerobic oxidationfrom a geochemical perspective are convincingŽIversen and Blackburn, 1981; Iversen and Jørgen-

.sen, 1985 . This methanotrophic process is particu-larly active in marine and brackish sediments, nor-mally at the base of the sulphate reduction zone. Thestrongest geochemical indications for anaerobic CH 4

Ž .oxidation are: 1 the dramatic decrease in CH 4

concentrations across the CH production–sulphate4Ž . Ž .reduction boundary Fig. 3 , 2 the preferential loss

of CH compared with higher molecular weight4Ž .hydrocarbons in the same location, and 3 the sys-

tematic shift in C and H isotope ratios of CH 4

consistent with the loss of CH in the consumption4Žzone e.g., Whiticar and Faber, 1986; Alperin et al.,.1988 .

Over the past 20 years studies of CH distribu-4

tions in marine sediments identified the diageneticzonation between sulphate reduction and CH for-4

Žmation e.g., Claypool and Kaplan, 1974; Martens.and Berner, 1977; Whiticar, 1982 . In the simplest of

terms, the concave downward shape of the depth


Žprofile of dissolved sulphate concentration Figs. 3.and 7a is the result of the balance between down-

ward diffusing sulphate, microbial uptake by SRBand vertical pore fluid advection. Analogously, theconcave upward shape of the CH concentration4

profile could not be maintained by diffusion andŽadvection mechanisms alone see Reeburgh, 1976;

.Martens and Berner, 1977 . Bacterial anaerobic oxi-dation at the base of the sulphate zone is responsiblefor active CH removal. However, complete con-4

sumption of CH does not occur and trace levels of4

CH are found in both sulphate-bearing and oxic4Ž .environments. Whiticar and Faber 1986 and other

Ž .investigators, including Martens and Berner 1977 ,Ž .Oremland et al. 1987 and Adams and van Eck

Ž .1988 , demonstrated that CH can persist in the4

presence of trace amounts of sulphate, approximately0.2 mM in the sulphate zone of marine, brackish andfreshwater sediments. Possible explanations for this

Ž . Ž .are: 1 restricted in situ methanogenesis, 2 in theseenvironments there is a CH concentration threshold4

below which CH is not attractive to methanotrophs,4Ž .or 3 that specific nutrients or co-metabolites are not

present. Methane buildup in the sulphate zone maybe related to consumption rates being lower than theinflux of CH . Vertical gas ebullition could rapidly4

introduce CH into the sulphate zone, or in certain4

circumstances the buildup could be due to inhibitionof CH oxidation.4

Bacterial consumption of CH appears to proceed4

at a significantly greater rate than for higher molecu-lar weight hydrocarbon gases such as ethane, propane

Ž . Ž .Fig. 12. Natural gas interpretative ‘‘Bernard’’ diagram after Bernard et al., 1978 combining the molecular and isotope compositionalˇinformation. Lines A and j; are calculated mixing lines for possible gas bacterial and thermogenic mixtures with end-member isotope

13 5 ˇŽ . � Ž .4 Ž . Ž .d C and molecular C r C qC compositions of y100‰, 10 ; y45‰, 2 A and y55‰, 5000; y45‰, 50 j; , respectively.CH 1 2 34

The relative compositional effects of migration or oxidation are also indicated.


or butanes. Thus, as CH is continually removed, a4

molecular fractionation occurs which relatively en-riches the residual hydrocarbon gas in the higherhomologues. The proportion of CH in a hydrocar-4

bon gas mixture can be expressed on a volumepercentage basis by the ‘‘Bernard parameter’’,

Ž . ŽC r C qC see Bernard et al., 1978; Whiticar,1 2 3.1990 . In CH formation zones which are devoid of4

Ž .thermogenic hydrocarbons, C r C qC is typi-1 2 3

cally 103 to 105. In the CH consumption zone the4Ž .C r C qC ratio can decrease to values of less1 2 3

than 10. Molecular ratios less than 50 are also typicalfor thermogenic hydrocarbon gases, as shown in Fig.12 so that CH oxidation can lead to difficulties in4

the interpretation of natural gas sources.Analogous to methanogenesis, the bacterial up-

take of CH is associated with a KIE that enriches4

the residual CH in the heavier isotope. This frac-4

tionation is most pronounced for the CH carbon4

isotopes, and in some cases, has been observed forthe hydrogen isotopes in CH . The magnitude of the4

carbon KIE during CH consumption is much lower4

than that associated with substrate uptake bymethanogens. Culture experiments and models have

Žbeen used to substantiate this compare Tables 1 and.3 .

Several investigators have demonstrated with aer-obic culture studies that 12 C–CH is preferentially4

removed during its oxidative consumption. The mag-Ž .nitude of the carbon isotope fractionation ´ inCŽculture experiments varies between 5 and 30 Table

.3 . The only determinations of the hydrogen isotopeŽ .fractionation factor for CH oxidation ´ in an4 H

Žaerobic culture study lay between 95 and 285 Cole-.man et al., 1981 .

Three models were developed by Whiticar andŽ .Faber 1986 to calculate the carbon isotope fraction-

ation of CH during its anaerobic consumption. These4Ž . Ž .models are termed: 1 residual methane, 2 C2q

Ž .enrichment and 3 methane–carbon dioxide parti-tion models. A fourth, a concentration gradient

Ž .model, was published by Alperin et al. 1988 . Thelatter were also able to model the hydrogen KIEs forCH oxidation at the same location.4

Ž .The first model residual methane uses the rela-tionship of d

13C to the CH concentration inCH 44

interstitial fluids, as CH is removed by bacterial4

oxidation the residual gas is continually enriched in

Table 3Magnitude of C and H isotope effects associated with methaneconsumption

Ž .A Methanotrophy: natural observations

Model names ´ Ć-methane D-methane

Whiticar and Faber, 1986Residual methane 4C enrichment 18 to 142qCH –CO 5 to 204 2

Alperin et al., 1988Concentration 9 148King et al., 1989 16 to 27Tyler et al., 1994 22Steudler and Whiticar, 1998 up to 5

Ž .B Methanotrophy: laboratory experiments

Aerobic cultures ´ Ć-methane D-methane

Silverman and Oyama, 1968 11Lebedew et al., 1969 16Zyakun et al., 1979 30Barker and Fritz, 1981 5 to 31Coleman et al., 1981 13 to 25 95 to 285Zyakun et al., 1984 7 to 27

Ž .the heavier carbon isotope fraction Fig. 13 . Thefractionated CH uptake is described by a closed-4

Ž .system Rayleigh function similar to Eq. 5 :

d13C sd

13C q´ ln 1yF , 14Ž . Ž .CH CH4 ,t 4 ,i

where d13C is the carbon isotope ratio for theCH 4,i

Žinitial methane pool prior to oxidation e.g., in the. 13main CH formation zone , d C is that at time4 CH 4,t

t. To account for variations of the initial CH con-4

centrations between different environments, the ini-tial CH concentration pool is normalized to 100%.4

The shift in d13C to more positive values as aCH 4

function of CH consumption is shown in Fig. 14.4

The best fit to the data was obtained with a carbonŽ .fractionation factor ´ of about 4. Larger values ofC

´ around 20, chosen to model the data, do notC

describe the system satisfactorily.Ž .The second model C enrichment uses the2q

Žsimultaneous change in both the molecular BsŽ . ŽC r C q C and isotope composition d C s1 2 3

13 .d C of the bacterial gas during CH consump-CH 44


Fig. 13. Residual methane model showing the shift in d13C toCH 4

more positive values with decreasing methane concentration dueto methane oxidation at the base of the sulphate-reducing zoneŽ .labeled CH Oxidation Zone . The residual methane is the per-4

centage of methane remaining compared with the methane con-Ž .centration in the main zone of methanogenesis 100% .

Ž .tion to calculate the isotope fractionation ´ ac-C

cording to

ln B y ln Bt i 3´ s q10C 3ln B y ln B r10 q d C yd CŽ . Ž .t i t i

15Ž .

where the subscripts i and t are for the initial andŽtemporal conditions, respectively see Whiticar and

Ž . Ž ..Faber 1986 for derivation of Eq. 10 . The compo-sitional shifts for a variety of marine, marsh and

Žbrackish sediments are shown in Fig. 15 a modified.version of Fig. 12 . The carbon isotope fractionation

factors vary from ´ s7 to 14, as indicated in theCŽfigure. Other data from the Antarctic Whiticar and

.Suess, 1990 gave lower ´ values between 1.8 andC

5. The explanation for the lower bacterial selectivity

for these samples is not clear, but it may be relatedŽ .to the cold sediment temperatures y1.48C .

As described in Section 5, the carbon isotopeŽ Ž ..separation between CH and CO Eq. 7 is fairly4 2

constant and indicative of the formation pathwayŽ .methane–carbon dioxide partition model . In con-trast, CH oxidation causes a clear, strong decrease4

Ž 13 13 .in the carbon isotope separation d C –d C ,CO CH2 4

as indicated in Fig. 8. Initially, the greater amountsof CO than CH typically present obscure the oxi-2 4

dation trend. However, towards the latter stages ofconsumption, the carbon isotope separation is ob-served to have ´ values between 5 and 25. FromC

the d13C , d

13C coexisting pairs it is not onlyCO CH2 4

possible to distinguish between the methanogenicpathways but also to evaluate CH consumption4Ž .Fig. 8 .

Ž .Alperin et al. 1988 determined the isotope shiftsin CH from anaerobic Skan Bay sediments as a4

Žfunction of the CH concentration gradient con-4.centration gradient model . By subtracting the possi-

ble effects of isotope fractionation associated withdiffusion, they were able to apply diagenetic equa-tions to estimate the magnitude of both the C and Hisotope effects for anaerobic CH oxidation. The4

carbon isotope fractionation factor ´ was found toCŽ .be 8. The hydrogen isotope fractionation factor ´ D

for the same samples was 146, which is consistent

13 Ž .Fig. 14. Relationship of d C to the percent % residualCH 4Ž Ž ..methane from bacterial methane consumption Eq. 14 . Due to

Ž .the low isotope fractionation factor ´ s4 a significant shift inCŽ .the methane carbon isotope ratio is first seen when most ca. 80%

of the methane has been oxidized.


Ž .Fig. 15. Modified ‘Bernard’ diagram after Bernard et al., 1978� Ž .4showing the combined shifts in molecular C r C qC and1 2 3

Ž 13 .methane carbon isotope d C ratios resulting from fraction-CH 4

ated bacterial consumption of methane in marine and brackishŽ . Ž .sediments. Carbon fractionation factors ´ based on Eq. 15 lieC

between 7 and 14.

with the values reported for aerobic cultures byŽ .Coleman et al. 1981 .

The possibility of aerobic methane consumptionŽ .in soils was recognized by Keller et al. 1983 and

Ž .Seiler et al. 1984 , but only more recently it hasbeen accepted as an important sink of atmospheric

Žmethane e.g., Steudler et al., 1989; Ojima et al.,. Ž . Ž .1993 . Steudler et al. 1989 , Mosier et al. 1991

and others have indicated that there is a close rela-tionship between methanotrophs and ammonia-oxidizing bacteria in soils. The carbon KIEs associ-ated with the soil uptake of methane have been

Ž . Ž .reported by King et al. 1989 , Tyler et al. 1994Ž . Ž .and Steudler and Whiticar 1998 Table 3 . The

former two, using chambers and incubations, respec-tively, found ´ values around 16 to 27.C – CO ,CH2 4

The latter, using in situ gas microsampling of soilgases at Harvard Forest found carbon isotope effectsfor the consumption to be significantly lower than5‰.

The range of C and H isotope effects from experi-mental and field observations are summarized inTable 3. Overall, it can be commented that carbonisotope fractionation during CH oxidation in anaer-4

obic sediments is less than in aerobic culture studies.In addition, methanotrophs appear to be less selec-tive between the lighter and heavier carbon isotopesthan the methanogens.

7. Conclusions

In combination, the C and H isotope signatures ofCH are frequently adequate to reliably characterize4

bacterial or thermogenic natural gas types. Situa-tions, such as mixing of different natural gases orwhere extreme substrate depletion and consumptionoccur, could produce ambiguous methane isotopesignals. In these cases, the C- and H-isotopes ofmethane, in concert with the coexisting isotope infor-mation on CO and H O, are excellent tracers of the2 2

processes of bacterial CH formation and consump-4

tion. The magnitudes of the KIEs are consistent andsufficiently large to generate significant isotope frac-tionations that are diagnostic for the various pro-cesses. A clear understanding of these effects isnecessary to relate the isotope signal of a natural gasto its source.

In view of the increasing atmospheric CH con-4Žcentration ca. 0.5%–1% annually in the lower tro-

.posphere and, as a radiatively active atmospherictrace gas having direct effect on global climatechange, it is important to attach precise isotopesignatures to the various CH sources. Stable iso-4

topes are an elegant method to differentiate thevarious fluxes and numerically constrain the magni-tudes of both CH emissions to and removal from4

the lower troposphere. In this way our comprehen-sion of geochemical and microbiological processesinvolved in natural gas habitats and compositionswithin the geosphere are the key to understandingatmospheric CH change.4

Acknowledgements

Collaboration with microbiologists, including R.Oremland, USGS; V. Muller and M. Blaut, Univ.¨Gottingen; N. Iversen, Univ. Aalborg; K. Stetter,Üniv. Regensburg and R. Conrad, Max Planck Inst.,Marburg have led to much of the understandingexpressed in this paper. This work has been funded,in part, through BMFT grants in Germany and byNSERC grant 105389 in Canada.


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