Lipid biomarker patterns of methane-seep microbialites from the Mesozoic convergent margin of California

Organic Geochemistry 37 (2006) 1289–1302

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OrganicGeochemistry

Lipid biomarker patterns of methane-seep microbialites fromthe Mesozoic convergent margin of California

Daniel Birgel a,*, Volker Thiel b, Kai-Uwe Hinrichs a, Marcus Elvert a,Kathleen A. Campbell c, Joachim Reitner b, Jack D. Farmer d, Jorn Peckmann a

a DFG-Research Center for Ocean Margins, University of Bremen, Postfach 330 440, D-28334 Bremen, Germanyb Center for Geosciences, University of Gottingen, Goldschmidtstr. 3, D-37077 Gottingen, Germany

c University of Auckland, Geology Department, Private Bag 92019, Auckland, New Zealandd Arizona State University, Geology Department, Tempe, AZ, USA

Received 5 September 2005; accepted 27 February 2006Available online 6 June 2006

Abstract

In order to reconstruct biogeochemical pathways at Mesozoic methane-seeps, a set of Late Jurassic (Tithonian) to EarlyCretaceous (Aptian/Albian), 13C-depleted seep-limestones from forearc strata in western California were subjected todetailed molecular-isotopic biomarker analyses. Two of the microbial carbonate deposits are turbidite-hosted/fault-related, whereas one is hosted in serpentinite in a diapir-related setting. The limestones contain 13C-depleted archaeal lipidbiomarkers such as crocetane (d13C � �80&) and PMI (� �100&), indicative of an involvement of anaerobic oxidationof methane (AOM) in carbonate precipitation. Isotopically depleted crocetane in the Tithonian sample represents the old-est reported occurrence of this compound at methane-seeps. In the set of samples, a series of strongly 13C-depleted, regularC21 to C24 isoprenoids possibly results from diagenetic alteration of archaeal sesterterpanylglycerol diethers as suggestedby the presence of the putative intermediate 3,7,11,15,19-pentamethylicosanoic acid. 13C-depleted 17a(H),21b(H) and17b(H),21a(H)-hopanes (C30–C34) with 22S- and 22R- isomer couplets (>C31) are present in all samples in distributionsindicative of a moderate thermal maturity. Low d13C values (�78& to �60&) suggest that these are derived from anaer-obic bacteria involved in AOM. Notably, 22S-isomers are consistently enriched in 13C relative to their 22R-counterparts.Our samples represent 70 myr of seepage activity and AOM along the Mesozoic margin of western California, filling thegap between the currently oldest methane-seep biomarker record from the Oxfordian (Late Jurassic) and the more widelyrecognised Cenozoic examples.� 2006 Elsevier Ltd. All rights reserved.

1. Introduction

In recent years, considerable interest has beendevoted to the study of modern and ancient meth-

0146-6380/$ - see front matter � 2006 Elsevier Ltd. All rights reserveddoi:10.1016/j.orggeochem.2006.02.004

* Corresponding author. Tel.: +49 421 218 65704/; fax +49 421218 65715.

E-mail address: [email protected] (D. Birgel).

ane-seeps. Modern hydrocarbon seepage preferen-tially occurs at geotectonic, geochemical andbiological interfaces, where methane is exiting theseafloor. Numerous studies have dealt with chemo-symbiotic invertebrate assemblages, sedimentaryfabrics, microbial mats, and carbon isotopes associ-ated with the microbial carbonates precipitatingwithin such environments (see Peckmann and Thiel,

.

1290 D. Birgel et al. / Organic Geochemistry 37 (2006) 1289–1302

2004; Campbell, 2006, for reviews). The key processat methane-seeps is the anaerobic oxidation ofmethane (AOM), which is performed by consortiaof sulphate-reducing bacteria (SRB) and methano-trophic archaea (Elvert et al., 1999, 2000; Hinrichset al., 1999, 2000; Boetius et al., 2000; Pancostet al., 2000; Orphan et al., 2001; Thiel et al.,2001). Although the exact pathway is still not clari-fied, it has been shown that the formation of car-bonates is caused by an increase in alkalinityresulting from the metabolic activity of the microbesinvolved in AOM (Ritger et al., 1987; Paull et al.,1992; Michaelis et al., 2002).

Numerous studies of modern methane-seep envi-ronments provide detailed information on specificlipid biomarker inventories associated with meth-ane-seep deposits (Hinrichs and Boetius, 2002; Pan-cost and Sinninghe Damste, 2003; Blumenberget al., 2004; Elvert et al., 2005). In both modernand ancient seep-deposits, however, microbial sig-nals reflecting the process of AOM are diluted byallochthonous inputs and/or influenced by biodeg-radation (Hinrichs et al., 2000; Peckmann et al.,2002). In addition, secondary migration of hydro-carbons and progressive alteration through thermalmaturation may obscure original signals fromancient AOM communities (Goedert et al., 2003).

Ancient methane-seep and hydrothermal-ventdeposits have been reported worldwide by variousauthors and are known to occur since the Protero-zoic eon (see Campbell and Bottjer, 1995; Campbell,2006). To identify and locate ancient methane-seeps,Campbell and Bottjer (1993) proposed a ‘seep-search strategy’ based on structural relationships,palaeontology, and stable carbon and oxygen isoto-pic compositions. From a comprehensive data set ofstable carbon and oxygen isotopes, Campbell et al.(2002) defined three stable isotope fields, wheremodern seep-carbonates are typified by high d18O-values and ancient carbonates show lower d18O-val-ues, including seep deposits from locations at ColdFork of Cottonwood Creek (CC), Paskenta (PSK),and Wilbur Springs (WS) discussed here. For thesethree California sites Campbell et al. (2002) sug-gested a mixed thermogenic/biogenic methanesource based on d13Ccarbonate signatures between�44& and �20&. With regard to WS, which ishosted in a sedimentary serpentinite, an involve-ment of carbon derived from oxidation of abiogenicmethane is another possibility that should be con-sidered. Serpentinization is thought to generatemethane in a Fischer–Tropsch type reaction (Kelley

and Fruh-Green, 1999; McCollom and Seewald,2001).

Lipid biomarker analysis of authigenic carbon-ates represents a powerful tool to identify and clas-sify AOM activity at methane-seeps (e.g., Peckmannand Thiel, 2004). Within the Mesozoic convergentmargin of western California, several methane-seepcarbonates have been recognised containing fossilsof seep biota with known descendants among livingchemosymbiotic groups Campbell and Bottjer(1993); Campbell, 2006. Although the prokaryoticAOM communities have attracted increasing atten-tion in recent years, and their lipid biomarker inven-tory has been well-examined from various sites allover the world, still rather little information is avail-able on molecular fossils from ancient seep deposits.To date, the majority of lipid biomarker reportshave dealt with ancient methane-seep occurrencesfrom Cenozoic strata (Peckmann et al., 1999,2002; Thiel et al., 1999, 2001; Burhan et al., 2002;Goedert et al., 2003; Peckmann and Thiel, 2004).Almost no biomarker records from Mesozoic andPalaeozoic methane-seep deposits are known sofar, except of one Jurassic (Oxfordian) deposit inFrance that contains isotopically depleted PMI(Peckmann et al., 1999). Here we report lipid bio-marker distributions in five samples from threeMesozoic sites, representing 70 myr of seepageactivity on the western North American convergentmargin. The good preservation of lipid biomarkerswithin the investigated limestones allows us todescribe past activity of chemosynthetic prokary-otes associated with ancient hydrocarbon seepage.

2. Materials and methods

2.1. Study area

Five limestone samples from three different loca-tions along the western margin of California havebeen studied (Fig. 1). A detailed description of thegeological and palaeontological context can befound elsewhere (see Campbell et al., 2002, and ref-erences therein).

Two of the locations, CC and PSK, are situatedwithin a turbidite-hosted/fault-bounded setting(Fig. 1). Such synsedimentary faults acted as flowpathways for hydrocarbon-charged fluids generatedwithin an accretionary prism and/or fluids fromgreater depths. The carbonates are patchily distrib-uted over a time span of at least 70 myr of forearc

Fig. 1. (A) Location map for Mesozoic seep-deposits (filled circles) of western California modified from Campbell et al. (2002). The threeseep-carbonate occurrences include the Cold Fork of Cottonwood Creek (CC), Paskenta (PSK), and Wilbur Springs (WS) localities. Asimplified geological overview shows a north-south trending convergent margin system, including from west to east: the FranciscanAccretionary Complex (Jurassic to Early Cretaceous) – broken formation and tectonic slivers of oceanic crust substratum; the GreatValley Group (Jurassic to Palaeogene) – forearc turbidites (siliciclastic); and the Sierra Nevada batholith – a Mesozoic volcanic arc. Blankareas indicate rock types not related to our study. (B) The CC site (Aptian–Albian) is situated close to the junction of the synsedimentaryCold Fork and Sulphur Springs fault. Val, Valanginian; H-B, Hauterivian–Barremian; Apt, Aptian; Alb, Albian; T, Tertiary. Geology andstratigraphic units modified from Bailey and Jones (1973). (C) The PSK location (Tithonian) is associated with a synsedimentary, listricfault zone. CRO, coast range Ophiolite; uJ, IK, late Jurassic, early Cretaceous. Geology modified from Jones et al. (1969). (D) The WSdeposit (Hauterivian) occurs within a diapir-associated sedimentary serpentinite (Kss) affiliated with the CRO. Kugv, Cretaceous,undifferentiated Great Valley Group turbidites. Simplified map from Carlson (1984).

D. Birgel et al. / Organic Geochemistry 37 (2006) 1289–1302 1291

subduction history of the Mesozoic era (Late Juras-sic, Tithonian to Early Cretaceous, Aptian/Albian).

The third seep location, WS, is situated in a dia-pir-associated sedimentary serpentinite, or foliateserpentinite breccia (Fig. 1). These rocks relate toan unusual, temporally and spatially restricted tec-tonic event that occurred during the Early Creta-ceous (Hauterivian to Albian) along the westernCalifornia margin (Campbell and Bottjer, 1993).The submarine serpentinite extrusion exposed at

WS represents an analogue to modern serpentinemud volcanoes and seep-carbonate chimneys found,for example, in the outer Marianas Basin (Haggerty,1991).

2.2. Sample preparation, lipid extraction and analysis

For lipid biomarker analyses, carbonates werecrushed to small pieces and cleaned by repeatedwashing with 10% HCl and acetone. Doubly dis-

1292 D. Birgel et al. / Organic Geochemistry 37 (2006) 1289–1302

tilled water was added to the samples and 10% HClwas slowly poured on the samples to dissolve thecarbonate. To avoid transesterification reactions atlow pH, no HCl was added after �80% of the car-bonate matrix had been dissolved. Remaining car-bonate pieces were removed and the residue wascentrifuged. After washing with water, the sampleswere subsequently saponified in 6% KOH in metha-nol. The supernatants were decanted and the resi-dues were extracted repeatedly by ultrasonicationin dichloromethane/methanol (3:1; v:v) until the sol-vents became colourless. The combined superna-tants were partitioned in dichloromethane vs.water treated with 10% HCl to pH 2. The organicextracts were dried with sodium sulphate and sepa-rated by column chromatography (Merck silica gel60, 0.063–0.200 mm; i.d.: 15 mm, length: 35 mm)into fractions containing (a) hydrocarbons (n-hex-ane), (b) alcohols/ketones (dichloromethane), (c)carboxylic acid fraction (dichloromethane/metha-nol, 2:1; v:v). Fatty acid methyl esters (FAMEs)were prepared from free fatty acids by subjectingthe dried fatty acid fraction to a mixture of trimeth-ylchlorosilane/methanol (1 ml, 1:8, v:v) in a screw-cap vial (2 h, 60 �C). The reaction mixture was driedunder nitrogen and the resulting methyl esters wereeluted from a short silica column with dichloro-methane. All fractions were examined by coupledgas chromatography–mass spectrometry using aThermo Electron Trace GC–MS equipped with a30-m DB-5MS fused silica capillary column(0.32 mm i.d., 0.25 lm film thickness). The carriergas was He. The GC temperature program usedwas as follows: injection at 60 �C, 2 min isothermal;from 60 to 150 �C at 15 �C min�1; from 150 to320 �C at 4 �C min�1; 20 min isothermal. Identifica-tion of individual compounds was based on com-parison of mass spectra and GC retention timeswith published data and reference compounds.Compound-specific carbon isotope analyses (irm-GC/MS) were carried out with a Thermo ElectronTrace GC coupled via a Thermo Electron GC-com-bustion-III-interface to a Thermo Electron Delta-plusXP mass spectrometer. GC conditions wereidentical to those described above. Carbon isotoperatios are given as d-values (d13C (&)) relative tothe V-PDB-standard and have been corrected forthe addition of carbon during preparation ofFAMEs. Several CO2-pulses of known d13C valueat the beginning and end of each run were usedfor calibration. Instrument precision was checkedusing a mixture of n-alkanes (n-C15 to n-C29) with

known isotopic composition. Standard deviationsare <0.4&.

3. Results

Gas chromatograms (C12+) of hydrocarbons andFAMEs were obtained from (1) CC (Aptian/Albian; samples CC-1, CC-60-3; CC-0-1), (2) WS(Hauterivian; sample WS-9), and (3) PSK (Titho-nian; sample PSK-1-8B).

3.1. Isoprenoids

In the hydrocarbon fraction, crocetane and co-eluting phytane are the most abundant isoprenoids.These compounds are accompanied by PMI andpristane (Fig. 2). Additional isoprenoid hydrocar-bons include farnesane and nor-pristane, and a ser-ies of tentatively identified regularpseudohomologues with 21–25 carbon atoms(Fig. 3). Furthermore, acyclic biphytane, togetherwith C38 and C39 pseudohomologues are presentat low concentrations in most samples. In the fattyacid fraction, isoprenoidal structures include phy-tanic acid and low amounts of 3,7,11,15,19-pentam-ethylicosanoic acid.

3.2. n-Alkanes

The detected n-alkanes range from C12 to C32. Auniform distribution was observed at all sites, super-imposed by increased concentrations of C14–C18

alkanes (Fig. 2). Another significant group of ali-phatic compounds are represented by iso- and ante-

iso-alkanes from C13 to C24, peaking at shorterchains from C14 to C18.

3.3. Fatty acids

Major acyclic compounds are fatty acids fromC12 to C30. The carbon number distribution is sim-ilar to that of n-alkanes, with increased concentra-tions of C14–C18, and maximising at C16 and C18

fatty acids (Fig. 4). For CC locations, C20–C30 fattyacids show a slight even-over-odd carbon numberpredominance. Iso- and anteiso-fatty acids (C12–C20) are abundant and peak at 14–18 atoms.

3.4. Cyclic terpenoids

The most abundant cyclic compounds are hopa-noids. In the hydrocarbon fractions, C27–C35

Fig. 3. Partial hydrocarbon fraction gas chromatogram (TIC) showing the distribution of isoprenoids from three seep-limestones asindicated. Black dots, n-alkanes; white triangles, iso- and anteiso-alkanes; grey triangles, regular isoprenoids; Cr, crocetane; Ph, phytane;PMI, 2,6,10,15,19-pentamethylicosane.

WS-9Hauterivian

Retention time

Rel

. int

ensi

ty

PSK-1-8BTithonian 23

PMI

Sq

Cr/Ph

Pr16

BpFN

Cr/Ph

FN

PMI

16Pr Sq

Bp

CC-60-3Aptian/Albian

23

PMI

Sq

Cr/Ph

Pr16

BpF N

23

IS

IS

IS

Fig. 2. Gas chromatograms (total ion current: TIC) of hydrocarbon fractions from CC, PSK and WS seep-limestones. Black dots, n-alkanes; small white triangles: iso-/anteiso-alkanes; grey triangles, isoprenoids; F, farnesane; N, nor-pristane; Pr, pristane; Ph, phytane; Cr,crocetane; PMI, 2,6,10,15,19-pentamethylicosane; Sq, squalane; Bp, biphytane, IS, internal standard.

D. Birgel et al. / Organic Geochemistry 37 (2006) 1289–1302 1293

hopanes were identified with 17a(H),21b(H)-30-nor-hopane and 17a(H),21b(H)-hopane being mostabundant. Among the extended hopanes (>C30),C31–C33 compounds predominate, and concentra-tions decrease towards the C35 pseudohomologue(Fig. 5). The ‘geological’ 17a(H),21b(H) stereo-

chemistry is the most prevalent configurationobserved. In addition, diagenetically formed C30–C32 17b(H),21a(H) isomers (moretanes) are presentin traces. In the fatty acid fraction, C31–C33

17a(H),21b(H)- and trace amounts of17b(H),21a(H)-hopanoic acids were found, with a

Fig. 4. Gas chromatograms (TIC) of fatty acid fractions from CC, PSK and WS seep-limestones. Black dots, fatty acids; small whitetriangles, iso- and anteiso-fatty acids; grey triangles, isoprenoidal acids; black diamonds, hopanoic acids; Ph, phytanoic acid.

Fig. 5. Partial gas chromatograms of hydrocarbon fractions showing the distribution of hopanes from three seep-limestones as indicated.Black dots, n-alkanes; grey triangles, isoprenoids; black squares, steranes; black triangles, 17a(H),21b(H)-hopanes (>C30 = 22S); whitetriangles, 17a(H),21b(H)-hopanes (>C30 = 22R); fasciated triangles, 17b(H),21a(H)-moretanes; dotted triangles, 17a(H)-22,29,30-tris-norhopane; G, gammacerane; Sq, squalane; Bp, biphytane; IS, internal standard.

1294 D. Birgel et al. / Organic Geochemistry 37 (2006) 1289–1302

strong preponderance of the C32 pseudohomologue(Fig. 4). Hopanes >C31 show isomerisation at theasymmetric C-22 atom, with 22S (geological) and22R (biological) epimers. The abundances of a,b-and b,a-hopanoids and (22S/22S + R)-ratios ofextended hopanes (PC31) provide information on

the thermal maturity of the samples that modifiedthe original lipid distribution (Peters et al., 2005).In addition, gammacerane was found as a minorcompound in the hydrocarbon fraction (Fig. 5). Tet-rahymanol (gammaceran-21a-ol), a triterpenol witha pentacyclic carbon skeleton closely related to the

Table 1d13C values (& relative to V-PDB) of various archaeal, bacterial, and eukaryotic biomarkers (hydrocarbon fraction) from Mesozoic seep-limestones, tr, trace amount

Compound Putative source Precursor Lipids CC-1 CC-60-3 CC-0-1 WS-9 PSK-1-8B

iso-C14 Bacteria Fatty acids �42.0 �44.1 �47.7 �57.5 �43.9 Straight-chain andbranched hydrocarbons(bacteria and eukaryotes)

anteiso-C14 Bacteria Fatty acids �50.0 �50.1 �52.9 �60.7 �48.1n-C14 Bacteria, eukaryotes Fatty acids �44.1 �48.2 �47.3 �47.6 �43.8iso-C16 Bacteria Fatty acids �44.0 �47.2 �48.0 �39.6 �45.6anteiso-C16 Bacteria Fatty acids �47.1 �48.7 �49.1 �47.8 �46.7n-C16 Bacteria, eukaryotes Fatty acids �46.0 �50.1 �48.9 �66.7 �42.9n-C18 Algae Fatty acids �44.9 �47.5 �47.0 �68.3 �42.9n-C26 Algae Leaf waxes, n-alkyl bearing lipids �42.9 �42.8 �41.9 n.d. �38.6n-C29 Higher land plants Leaf waxes, n-alkyl bearing lipids n.d. �37.5 �36.8 n.d. �37.6

Phytane/crocetane (mixture) Archaea and Algae Crocetane and unsaturatedderivatives/archaeol derivatives

�75.6 �87.2 �91.7 �98. 4 �46.8 Acyclic isoprenoids(archaea)

reg. C21isoprenoid Archaea ? reg. PMI derivatives/sesterterpanyl diethers

�60.9 �79.7 �111.8 �78.0 �49. 0

reg. C22isoprenoid Archaea ? reg. PMI derivatives/sesterterpanyl diethers

�52.6 �111.2 �117.4 tr tr

reg. C23isoprenoida Archaea ? reg. PMI derivatives/sesterterpanyl diethers

�51.6 �76.6 �85.9 tr tr

reg. C24isoprenoid Archaea ? reg. PMI derivatives/sesterterpanyl diethers

�72.4 �75.5 �79.8 tr tr

PMIb Archaea Unsaturated PMI derivatives/sesterterpanyl diethers

�88.8 �91.1 �102.1 �100.8 �121.8

C40-biphytane Archaea GDGT’s �82.4 �73.5 �87.4 �105.1 �100. 8

17a(H),21b(H)-hopane Bacteria Hopanepolyols �53.4 �58.4 �53.6 �63. 6 �60.0 Pentacyclic triterpenoids(bacteria)17b(H),21a(H)-moretane Bacteria Hopanepolyols �67.9 �66.3 �68.1 tr tr

17a(H),21b(H)-31-hopane (22S) Bacteria Hopanepolyols �58.4 �61.0 �61.8 �61.5 tr17a(H),21b(H)-31-hopane (22R) Bacteria Hopanepolyols �57.3 �63.2 �61.9 �68.4 tr17b(H),21a(H)-31-moretane (22S/R ?) Bacteria Hopanepolyols �65.9 �68.5 �69.8 tr tr17a(H),21b(H)-32-hopane (22S) Bacteria Hopanepolyols �54.0 �60.7 �62.3 tr tr17a(H),21b(H)-32-hopane (22R) Bacteria Hopanepolyols �69.2 �73.7 �77.8 tr tr17a(H),21b(H)-33-hopane (22S) Bacteria Hopanepolyols �59.8 �63.3 �65.3 tr tr17a(H),21b(H)-33-hopane (22R) Bacteria Hopanepolyols �61.2 �73.5 �73.5 tr trGammacerane Ciliates Tetrahymanol �65.9 �68.5 �69.8 tr tr

a Co-elution of regular C23-isoprenoid and anteiso-C21-alkane.b Putative mixture of regular and irregular isomers of PMI.

D.

Birg

elet

al.

/O

rga

nic

Geo

chem

istry3

7(

20

06

)1

28

9–

130

21295

Table 2Carbon isotopic compositions (& relative to V-PDB) of various archaeal, bacterial, and eukaryotic biomarkers (fatty acid fraction) fromMesozoic seep-limestones, tr, trace amount

Compound Putative source CC-1 CC-60-3 CC-0-1 WS-9 PSK-1-8B

n-C14 SRB, diverse marine sources �40.9 �39.8 �42.0 �52.1 trn-C16 SRB, diverse marine sources �38.5 �37.6 �42.4 �50.5 trn-C18 SRB, diverse marine sources �30.8 �34.0 �31.5 �40.6 trn-C26 Higher land plants �39.9 �38.6 �39.2 �41.4 triso-C15 Bacteria �41.1 �43.8 �40.5 �38.0 tranteiso-C15 Bacteria �54.2 �47.8 �46.6 �44.4 triso-C17 Bacteria �46.1 �46.1 �53.7 tr tranteiso-C17 Bacteria �50.3 �45.0 �58.8 tr trPhytanoic acid Archaea and algae �76.5 �79.2 �76.6 �62.5 tr17a(H),21b(H)-32-hopanoic acid Bacteria �47.1 �40.6 �48.6 tr tr17b(H),21a(H)-32-hopanoic acid Bacteria �47.9 �40.5 �47.3 �48.1 tr

1296 D. Birgel et al. / Organic Geochemistry 37 (2006) 1289–1302

hopanoids is the putative precursor of gammace-rane (ten Haven et al., 1989).

3.5. Compound specific stable carbon isotopes

Carbon isotopic compositions of selected bio-markers are given in Tables 1 and 2. Carbon isotopevalues vary from average marine (d13C: �35& to�30&) to extremely low 13C signals (<�100&).Lowest d13C values were determined for PMI(�122& to �88&), mixed phytane/crocetane(�98& to �47&) and for all other isoprenoids(�112& to �52&). Hopanoids typically had inter-mediate d13C values ranging from �70&

(17a(H),21b(H)-dihomo-hopane) to �40&

(17a(H),21b(H)-dihomo-hopanoic acid). Remark-ably, the ‘geological’ 22S hopane isomers (�65&

to �58&) were generally less depleted in 13C rela-tive to the ‘biological’ 22R isomers (�77& to�57&). Compared with isoprenoids and hopa-noids, n-alkanes are usually enriched in 13C(�45& to �35&).

4. Discussion

4.1. Post-depositional and alteration processes

4.1.1. General remarks

Biomarker inventories of ancient seep-limestonesmay have been affected by contributions of organicmatter from allochthonous sources and by biodegra-dation. Furthermore, migration of petroleum hydro-carbons and alteration by thermal maturation mayhave a strong post-depositional impact on the distri-bution, preservation and carbon isotopic composi-tion of biomarkers (e.g., Sinninghe Damste et al.,1995a; Goedert et al., 2003). In methane-rich envi-

ronments, carbonate precipitation fuelled by AOMmay lead to early entombment and fossilisation ofassociated organisms and appears crucial for thepreservation of organic compounds (e.g., Thielet al., 1999; Aloisi et al., 2002; Peckmann et al., 2002).

4.1.2. Thermal alteration

The limestones studied contain numerousorganic compounds that show no alteration com-pared to those extracted from modern methane-seeps. On the other hand, many of the ancient bio-markers are considered to be breakdown productsof known or unknown precursor lipids. From thebiomarker patterns it appears that unsaturated mol-ecules have been destroyed, incorporated into kero-gen, or hydrogenated. Carbon–carbon bonds alsomay have been cleaved. A commonly used thermalmaturity parameter is the extended hopane isomeri-sation index, calculated as 22S/(22S + 22R). Mostlocations show isomerisation of the C31–C35

pseudohomologues with 22S/(22S + 22R) ratiosranging from 0.55 to 0.60. Such values are reachedin the early oil window stage (Peters et al., 2005).However, an exception with considerably lowerthermal maturity is indicated by 22S/(22S + 22R)ratios of 0.43–0.53 for CC-0-1. Moreover, the pres-ence of moretanes, albeit in low concentrations,indicates that studied samples did not enter a latecatagenetic stage (cf., Koster et al., 1997; Peterset al., 2005).

4.2. Archaeal and bacterial lipid biomarkers inMesozoic authigenic carbonates

4.2.1. Isoprenoids

Characteristic archaeal compounds such asarchaeol, hydroxyarchaeol, and glycerol dialkyl

D. Birgel et al. / Organic Geochemistry 37 (2006) 1289–1302 1297

glycerol tetraethers (GDGT) (e.g., Teixidor et al.,1993; Hinrichs et al., 1999; Pancost et al., 2001; Blu-menberg et al., 2004), which predominantly derivefrom methanogens and methanotrophs, are onlypoorly preserved in thermally altered samples.Although the overall preservation of biomarker lip-ids under moderate maturity conditions appearsgood, cyclic biphytane carbon skeletons are morereadily thermally degraded and therefore not pre-served. Artificial maturation experiments on biphy-tane-containing sediments showed the absence ofcyclic biphytanes in samples with extendedhopane-22S/(22S + 22R) ratios >0.3, whereas acy-clic biphytanes were still present (Koopmans et al.,1996; Schouten et al., 1998). This situation mayapply to the samples investigated, where free acyclicbiphytane derivatives were determined in significantamounts (Fig. 5). The identified acyclic biphytanesshowed low d13C values (�105& to �72&), indicat-ing methanotrophic archaea as source organisms(Table 1). In modern methane-seep settings,GDGT-derived acyclic and cyclic biphytanes wereobserved with d13C as low as �96& (Pancostet al., 2001; Thiel et al., 2001).

Along with the 13C-depleted biphytanes, PMIand crocetane were found (Fig. 3). d13C values ofPMI range from �122& to �88& in samples fromthe CC and PSK localities (Table 1). In comparisonwith these limestones, the values found for the ser-pentinite-hosted WS sample did not show higherd13C values (PMI: �100.8&; Ph/Cr: �98.4&; Table1), not agreeing with a significant contribution fromabiogenic methane. Originally, PMI was identifiedin methanogenic and thermoacidophilic archaea,and has been widely used as a biomarker forarchaea (Holzer et al., 1979; Tornabene et al.,1979; Risatti et al., 1984; Schouten et al., 1997;Summons et al., 1998). Isotopically depleted PMI,crocetane, and their unsaturated analogues are fre-quently found at modern methane-seeps and arecommonly used as biomarkers for methanotrophicarchaea (e.g., Elvert et al., 1999, 2000; Thiel et al.,1999; Hinrichs et al., 2000; Michaelis et al., 2002).In many ancient samples as old as Oxfordian (Juras-sic), 13C-depleted PMI has been found as a persis-tent marker for AOM (Peckmann and Thiel,2004). 13C-depleted crocetane has not been reportedfrom seep-limestones older than Cenozoic. It iswidely agreed that methanotrophic archaea aresource organisms of 13C-depleted crocetane (seePeckmann and Thiel, 2004 and references therein).If present in ancient seep-carbonates, crocetane is

always accompanied by PMI and is similarlydepleted in 13C. However, at some modern seep-sites, PMI is abundant whereas crocetane is missing.This variation may indicate different archaea syn-thesising either both compounds or only PMI(Elvert et al., 2000; Thiel et al., 2001; Blumenberget al., 2004).

Formation of crocetane from the degradation ofPMI was recently proposed by Barber et al. (2001),who reported a pseudohomologous series of regularand irregular C20–C25 isoprenoids in Palaeozoic andMesozoic crude oils. Regular C20–C25 isoprenoidswere interpreted as diagenetic products of glyceroldiether lipids derived from halophilic or otherarchaea (Grice et al., 1998). In our samples, noirregular isoprenoids other than crocetane andPMI occur, while regular isoprenoids include farne-sane, nor-pristane, pristane, phytane, and a series ofC21–C24 compounds (Fig. 3). An early biosyntheticsource of these compounds and their incorporationinto authigenic carbonates is suggested by highabundances of crocetane, PMI, and their unsatu-rated derivatives in modern to sub-recent meth-ane-seep deposits (Elvert et al., 2001).

Archaeol (bis-O-phytanylglycerol), and itsbyproduct phytanol are biosynthesised by methano-trophic archaea related to the order Methanosarci-

nales and represent putative precursors of 13C-depleted phytane and shorter derivatives. This issupported by maturation experiments on methano-gens containing isoprenoid glycerol diethers andPMI (Rowland, 1990). After hydrous pyrolysis,only regular acyclic isoprenoids 6C20 were formed,all of which were produced from phytanyl diethers(Rowland, 1990).

Regular C20–C24 isoprenoids (Fig. 3) may bederived from thermal cleavage of ether-bonds fromprecursor glycerol diethers with C20 and C25 regularisoprenoid moieties containing at least one ses-terterpanyl chain, as previously suggested by Griceet al. (1998); (see also Hinrichs et al., 1999; Barberet al., 2001; Peckmann and Thiel, 2004). Such com-pounds are known to be synthesised by halophilicarchaea (Teixidor et al., 1993), but are also knownfrom modern methane-seep environments (Hinrichs,unpublished results). Strong 13C-depletions indicatea methanotrophic archaeal source. Therefore,AOM-related archaea need to be considered as a fur-ther source of sesterterpanyl lipids in addition to hal-ophilic archaea. Regular PMI, which was expected toco-occur with the regular C21–C24 isoprenoids, wasonly ambiguously identified in our samples, possibly

1298 D. Birgel et al. / Organic Geochemistry 37 (2006) 1289–1302

due to co-elution with the more prominent irregularPMI isomer (cf., Greenwood and Summons, 2003).A potential role of sesterterpanyl lipids as precursorsof regular isoprenoids is consistent with the occur-rence of 3,7,11,15,19-pentamethylicosanoic acid inCC samples (Fig. 6).

A further source of isoprenoids in our samplesmay be thermal degradation of ether lipids contain-ing biphytane moieties. Maturation experiments onarchaea containing 80% polar phytanyl- andbiphytanyl glycerol-phosphates generated regularC15–C25 isoprenoids and head-to-head C37, C39,and C40 isoprenoids after pyrolysis experiments(Rowland, 1990). Likewise, in the Mesozoic seep-carbonates, we observed biphytane along with aC39 head-to-head isoprenoid, both in moderateabundances and similar low d13C values (Fig. 5,Table 1). These compounds may therefore originatefrom a similar degradation pathway as proposed forthe C21–C24 isoprenoids.

4.2.2. Cyclic terpenoids

4.2.2.1. Hopanoids. The d13C values of the ab- andba-hopanes reported here (�78& to �60&, Table

Fig. 6. (A) Partial gas chromatogram (TIC) of fatty acid fractionshowing 3,7,11,15,19-PMI-acid from limestone sample CC-60-3;black dots, fatty acids. (B) Mass spectrum and fragmentationscheme of 3,7,11,15,19-PMI-acid.

1) provide evidence for an incorporation of meth-ane-derived carbon into the biomass of the sourcebacteria. Whereas biologically produced extendedhopanoids carry a 22R configuration, thermal mat-uration gradually leads to a mixture of 22R and 22S

diastereomers, with a maximum 22S/(22S + 22R)ratio of around 0.60 reached at the early oil windowstage (see Peters et al., 2005, and references therein).The results found in the Californian seep-limestonesrepresent values close to the isomerisation equilib-rium point, except for location CC-0-1, where lowerratios (0.44–0.53) were found. In all samples, how-ever, the 22R isomers are considerably moredepleted in 13C than their corresponding 22S iso-mers (Fig. 7). Such an isotopic distinction has beenobserved by Sinninghe Damste et al. (1995a) duringartificial maturation experiments. Gradual heatingof a mixture of two extended 22R hopenes with dif-ferent initial d 13C values resulted in an explicit d13Cvariation of the 22S and 22R isomers generated.After reaching the isomerisation equilibrium forhopenes (52% 22S), d13C values were identical forboth isomers. Accordingly, the isotopic differencesbetween the C-22 isomers suggest that equilibriumhas not been reached.

Hopanoic acids, together with bacteriohopane-polyols (BHPs) and hopanols, are the most abun-dant hopanoids in immature and modernsediments (Innes et al., 1997, 1998). With increasingmaturity, however, functionalised hopanoids arelacking or are present only in low concentrations(Jaffe and Gardinali, 1990). The d13C values forthe 17a(H),21b(H)-dihomo-hopanoic acid in thestudied carbonates vary from �48& to �41&, thusbeing noticeably higher than those of the hopanes(as low as �78&). Therefore it appears, that thehopanoic acids and hydrocarbons in the Californiaseep-carbonates derive from different sources (seealso Farrimond et al., 1998). Indeed, liberation ofextended hopanes in thermally mature samples isachieved typically by cleaving side-chains of BHPsbound in macromolecules (Koster et al., 1997; Far-rimond et al., 1998). During that process, distincthopanoid pseudohomologues originate from certainpolyfunctionalised side chains: C30-hopanes frombacteriohopanehexols, C31-hopanes from bacterio-hopanepentols, and C32-hopanes from bacterioho-panetetrols (Innes et al., 1997).

Isotopically depleted hopanoids have recentlybeen described from various methane-rich environ-ments (Elvert et al., 2000, 2001; Pancost et al.,2000; Werne et al., 2002; Thiel et al., 2003). These

08-

07-

06-

05-

1-CCpA ti /na Albi na

-06-CC 3pA ti /na Albi na

1-0-CCpA ti /na Albi na

-SW 9aH tu naivire

δ13C

[‰]

04-egarevA

C 13 S-

C 13 R-

C 23 S-

C 23 R-

C 33 R-

2~ ‰

41~ ‰

7~ ‰

6- 0‰

6- 2‰

5- 9‰

7- 3‰

6- 2‰

6- 9‰

C 33 S-

C 23 S- napoH oi ca c di

Fig. 7. d13C ratios of extended 17a(H),21b(H)-hopanes and 17a(H),21b(H)-32-hopanoic acid derived from four limestone samples. In theright box, averaged isotope ratios for single isomers (22S and 22R) from all measured samples are shown. The isotopic values in boldletters are representing the average isotopic difference between S- and R-isomers.

D. Birgel et al. / Organic Geochemistry 37 (2006) 1289–1302 1299

compounds predominantly comprise C30-hopanoidssuch as diplopterol and diploptene (Pancost et al.,2000; Thiel et al., 2001), which are known to be syn-thesised de novo by various aerobic bacteria, includ-ing methanotrophs (Rohmer et al., 1984). Inaddition, extended hopanoids, namely dihomo-hopanoic acid and dihomo-hopanol have been found(Pancost et al., 2000; Thiel et al., 2003). Onlyrecently, Pancost et al. (2005) found homo- anddihomo-hopanols after side-chain cleavage of BHPsextracted from seep-carbonates from the Gulf ofMexico. An AOM-related bacterial source for 13C-depleted hopanoids in seep deposits has been sug-gested (Elvert et al., 2000; Pancost et al., 2000,2005; Thiel et al., 2003), but until very recently, nostrict anaerobes producing hopanoids (e.g., sulphatereducers) were known. New support for the ideathat 13C-depleted hopanoids in AOM-environmentscould derive from anaerobic bacteria came from therecent identification of BHPs in enrichment culturesof strictly anaerobic planctomycetes (SinningheDamste et al., 2004), and cultures of Geobacter spe-cies (Fischer et al., 2005; Hartner et al., 2005). It istherefore possible that at least some of the hopa-

noids in the California seep-limestones derive tosome extent from anaerobic bacteria participatingin the cycling of methane-derived carbon.

4.2.2.2. Gammacerane. The biological precursor ofgammacerane, tetrahymanol, is produced by multi-ple groups of organisms including ferns, fungi, pho-tosynthetic bacteria, and marine ciliates, and isubiquitously observed in marine sediments (Zanderet al., 1969; Kemp et al., 1984; Kleemann et al.,1990; Harvey and McManus, 1991; Harvey et al.,1997). Recently, tetrahymanol, putatively producedby bacterivorous ciliates, has been described inmethane-seep sediments from a Mediterraneanmud volcano (Werne et al., 2002). In that environ-ment, d13C values of tetrahymanol varied from�38& at the sediment–water–interface to �80&

in the zone of AOM. Therefore, 13C-depleted gam-macerane found in the ancient seep-limestones canplausibly be interpreted as a biomarker for hetero-trophic organisms of the second trophic level (e.g.,Sinninghe Damste et al., 1995b; Werne et al.,2002; Pancost and Sinninghe Damste, 2003) thatfed on AOM-related bacteria or archaea.

1300 D. Birgel et al. / Organic Geochemistry 37 (2006) 1289–1302

5. Conclusions

• d13C-depleted archaeal and bacterial biomarkersin the Mesozoic limestones confirm that carbon-ate precipitation resulted from anaerobic oxida-tion of methane.

• Circumstantial evidence suggests that series ofstrongly 13C-depleted, regular isoprenoid hydro-carbons are derived from biphytanyl, sesterterpa-nyl, and biphytanyl di- and tetraethers viaputative intermediates (phytanic acid, PMI acid).

• Similar to modern seep-environments, isotopi-cally depleted hopanoids are abundant. Their dis-tribution is consistent with moderate thermalmaturation.

• d13C values of microbial biomarkers in serpenti-nite-hosted Wilbur Springs seep-deposit suggestthat abiogenic methane was not an importantcarbon source.

Acknowledgements

We thank Michael Bottcher and Steven Bouil-lon for editorial work and Richard Pancost(Bristol) as well as an anonymous reviewer forhelpful comments on the manuscript. Financialsupport was provided by the ‘Deutsche Fors-chungsgemeinschaft’ through the DFG-ResearchCenter for Ocean Margins, Bremen (RCOMContribution 0362) and Grant PE 847/4. KACcollected materials during a US National Re-search Council Associateship, administeredthrough NASA Ames Research Center; draftingsupport by L. Cotterall, University of Auckland,Geology Department.

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