Sedimentary Geology 263–264 (2012) 174–182

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Sedimentary Geology

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Tracing organic compounds in aerobically altered methane-derived carbonate pipes(Gulf of Cadiz, SW Iberia)

Raúl Merinero a,⁎, Marta Ruiz-Bermejo b, César Menor-Salván b, Rosario Lunar a, Jesús Martínez-Frías b

a Departamento de Cristalografía y Mineralogía, Facultad de Ciencias Geológicas, Universidad Complutense de Madrid, Avda Complutense s/n, 28040, Madrid, Spainb Centro de Astrobiologia, CSIC/INTA, associated with the NASA Astrobiology Institute, Ctra de Ajalvir km. 4, 28850, Madrid, Spain

⁎ Corresponding author. Tel.: +34 1 3944959; fax: +E-mail address: rmeriner@geo.ucm.es (R. Merinero)

0037-0738/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.sedgeo.2011.09.011

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 December 2010Received in revised form 12 September 2011Accepted 15 September 2011Available online 22 September 2011

Keywords:Gulf of CádizAerobic degradationCarbonate pipesLipids

The primary geochemical process at methane seeps is anaerobic oxidation of methane (AOM), performed bymethanotrophic archaea and sulfate-reducing bacteria (SRB). The molecular fingerprints (biomarkers) ofthese chemosynthetic microorganisms can be preserved in carbonates formed through AOM. However, thermalmaturity and aerobic degradation can change the original preserved compounds, making it difficult to establishthe relation between AOM and carbonate precipitation. Here we report a study of amino acid and lipid abun-dances in carbonate matrices of aerobically altered pipes recovered from the seafloor of the Gulf of Cadiz (SWIberian Peninsula). This area is characterized by a complex tectonic regime that supports numerous cold seeps.Studies so far have not determined whether the precipitation of carbonate pipes in the Gulf of Cadiz is a purelychemical process orwhethermicrobial communities are involved. Samples from this site show signs of exposureto oxygenatedwaters and of aerobic alteration, such as oxidation of authigenic iron sulfides. In addition, the deg-radation index, calculated from the relative abundance of preserved amino acids, indicates aerobic degradationof organic matter. Although crocetane was the only lipid identified from methanotrophic archaea, the organiccompounds detected (n-alkanes, regular isoprenoids and alcohols) are compatible with an origin from AOMcoupled with bacterial sulfate reduction (BSR) and subsequent aerobic degradation. We establish a relationamong AOM, BSR and pipe formation in the Gulf of Cadiz through three types of analysis: (1) stable carbonand oxygen isotopic composition of carbonate minerals; (2) carbonate microfabrics; and (3) mineralogicalcomposition. Our results suggest that carbonate pipesmay form through a process similar to the precipitation ofvast amounts of carbonate pavements often found at cold seeps. Our approach suggests that some organiccompound patterns, in combination with additional evidence of AOM and BSR, may help indicate the sourceof altered methane-derived carbonates commonly occurring in ancient and modern deposits.

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1. Introduction

At submarine methane seeps, groups of archaea and bacteria carryout anaerobic oxidation of methane (AOM) and sulfate-dependentanaerobic oxidation of methane (AOM) (e.g., Hinrichs et al., 1999;Boetius et al., 2000; Orphan et al., 2001; Reitner et al., 2005). AOM in-creases alkalinity, inducing the precipitation of authigenic carbonates(Hovland et al., 1987; Ritger et al., 1987; Paull et al., 1992). Molecularfingerprints (biomarkers) of these chemosynthetic microorganismsare well preserved in carbonate matrices, the characterization ofwhich is a useful tool to establish the relation between carbonate pre-cipitation andmethane seepage (Peckmann and Thiel, 2004). However,post-depositional processes such as biodegradation and thermal mat-uration alter the biomarker inventory of the carbonates, obscuringthe information they provide (Goedert et al., 2003; Birgel et al.,

2006) and making it difficult to establish the role of methane andmicrobial activity in carbonate formation.

Methane-enriched fluid venting is a widespread process on thecontinental slope of the Gulf of Cadiz, as reflected in the abundanceof methane-related seafloor structures in this area. These structuresinclude pockmarks (Baraza and Ercilla, 1996; Casas et al., 2003);mud volcanoes, some of which contain gas hydrates (Pinheiro etal., 2003; van Rensbergen et al., 2005; Niemann et al., 2006); andcarbonate-mud mounds bearing cylindrical carbonate deposits orchimneys, crusts and slabs (León et al., 2006). Biomarkers of methano-trophic archaea have been isolated and identified on carbonate crustsand pavements collected on mud-volcanoes of the Gulf of Cadiz(Niemann et al., 2006; Stadnitskaia et al., 2008). Previous studieshave used the term “chimneys” to refer to carbonate depositswith cylindrical or conical shapes (Díaz-del-Río et al., 2003). Thisterm implies a vertical channel that projects into the water columnduring its formation. However, it is more likely that these carbon-ate deposits formed in the sediment and were later shaped by ero-sion, making the term “pipe” more appropriate.

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Early studies suggested that the most probable origin of carbonatepipes is AOM coupled with sulfate reduction, but explicit evidence isstill lacking (Díaz-del-Río et al., 2003; León et al., 2006, 2007).Moreover,the possibility of purely chemical precipitation of the carbonate pipes,without a microbial contribution, cannot be excluded due to the com-plex tectonic regime governing the Gulf of Cadiz (Maldonado et al.,1999) and the evidence of hydrothermal imprints inmud-volcanofluidsin the Gulf of Cadiz (e.g. Hensen et al., 2007). We consider these argu-ments necessary in order to exclude processes other than methane oxi-dation as possible origins of carbonate pipes. Thiswould be an importantcontribution of our study, since previousworks on tubular carbonate de-posits (chimneys) in the Gulf of Cadiz have not clarified this question.

Previous studies of the carbonate pipes in the Gulf of Cadiz showedthat they were lying over the seafloor, exposed to oxygenated waters(Díaz-del-Río et al., 2003). More recently, Merinero et al. (2008)reported the oxidation of iron sulfides and pseudomorphic transforma-tion into iron oxyhydroxides. Thus, aerobic degradation of organicmatter originally preserved inside the pipes was expected, whichwould make it difficult to establish the relation between carbonatepipe formation and AOM by identifying biomarkers from methano-trophic microbes.

Here we analyze a large collection of cylindrical carbonate pipesfrom the Gulf of Cadiz using a combination of geochemistry (analysisof organic compounds and 13C and 18O isotopic composition of car-bonate minerals), mineralogy and petrology. The objectives of this re-search were to (1) determine the degree of aerobic alteration of thepipes; (2) verify the expected relation between AOM and the originof the pipes; and (3) characterize the organic compounds preservedwithin the pipes and establish their possible relation with aerobicdegradation of biomarkers from chemosynthetic microorganisms.

2. Area descriptions, materials and methods

2.1. Geological settings

The Gulf of Cadiz is located at the trending segment of theEurasian-African plate boundary that extends from the Azores to the

Fig. 1. Bathymetric map of the Gulf of Cadiz (modified from Merinero et al., 2009) showing tCornide mound, AR = Arcos mud-volcano, CO = Coruña mud-volcano). Locations of the m

Mediterranean Sea. The segment, referred to as the Gibraltar Arc, liesbetween the Gloria Fault and the western end of the Betic-Rifean oro-genic belt (Fig. 1). This area shows a complex geodynamic historywith various extensional stages, strike-slip and compression associatedwith the closure of the Tethys, the opening of theNorthAtlantic, and theAfrican-Eurasian convergence since the Cenozoic (Maldonado et al.,1999). Water exchange between the Mediterranean and Atlantic inthe region of the Strait of Gibraltar has been a crucial factor in the devel-opment of various mesoscale physical structures characteristic of theAtlantic Ocean seafloor (Criado-Aldeanueva et al., 2006; García-Lafuente et al., 2006; Mulder et al., 2006). Hydrocarbon-rich fluids aretrapped in a thick sedimentary formation called the Olistostrome orAllochthonous Unit of the Gulf of Cadiz (Medialdea et al., 2004). Thisformation emplaced during theMiddle Miocene and consists of Triassicevaporites and red beds and blocks of Cretaceous to Paleogene lime-stones (Maldonado et al., 1999). Migration and subsequent seepage ofthe fluids have occurred because of diapirism and a compressional re-gime, which affect the Olistostrome in response to the Africa-Eurasiaplate convergence (Maldonado et al., 1999; Medialdea et al., 2009). Asa consequence of these processes, many types of seafloor structuresformed (León et al., 2006): pockmarks (Baraza and Ercilla, 1996; Casaset al., 2003); mud-volcanoes (Pinheiro et al., 2003; van Rensbergenet al., 2005; Niemann et al., 2006); and carbonate-mud mounds bearingcylindrical carbonate deposits, crusts and slabs (Díaz-del-Río et al., 2003;Merinero et al., 2008). The close relationship observed between tectonicstructures and these hydrocarbon-derived features suggests that fluidventing is triggered by the formation of pressurized compartmentsbeneath thrust structures, which provide conduits for hydrocarbon-enriched fluids (Maestro et al., 2003; León et al., 2007).

2.2. Material recovery and data acquisition

Samples were taken from the Iberian continental margin of theGulf of Cadiz during the oceanographic cruises Anastasya 2000 and2001 aboard the research vessel Cornide de Saavedra. The study areawas extensively surveyed with swath bathymetry, multi-channeland ultra-high-resolution seismic reflection, gravimetry, magnetism,

he locations of the four sites where samples were collected (IB = Iberico mound, CN =ud-volcanoes and mud-mounds are from León et al. (2006) and González et al. (2009).

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underwater cameras, dredging, and gravity coring. A large number ofmud volcanoes, mud ridges, crater-like pockmarks, and large sedimentslides were mapped. The carbonate pipes were discovered during theAnastasya 2000 cruise while dredging an 870 m-deep, 120 m-tall car-bonate mound known as Iberico (Díaz-del-Río et al., 2003). Mostpipes were collected along or in close proximity to the main channelsof the Mediterranean Outflow Water using rectangular benthic-typedredges.More than200 carbonate pipeswere recovered at depths rang-ing from 850 to 1100 m during the cruises in 2000 and 2001. Here westudied 13 carbonate pipes from four different sites (Table 1).

2.3. Mineralogy, geochemistry and stable C and O isotope composition

We examined each specimen as a hand sample and in thin section(transversal and longitudinal views). From these slabs, we preparedthin sections for standard petrographic analysis. We made SEM andmicroprobe analyses on carbon-coated thin sections using a PhilipsXL20 scanning electron microscope with accelerating voltages of20–30 kV. We determined the carbonate mineralogy by X-ray diffrac-tion (XRD) using a Philips PX-1710 diffractometer operating at 40 kV,equipped with a graphite monochromator and an automatic diver-gent slip. We also analyzed the carbonate chemical composition byelectron microprobe (JEOL Superprobe JXA-8900M).

For isotopic measurements, samples were ground to b200 meshand reacted with 100% phosphoric acid at 25 °C for 3 h in the caseof calcite and at 25 °C for 3 days in the case of dolomite and ankerite.We extracted carbon dioxide from the carbonate samples accordingto the method of Al-Aasm et al. (1990) using a Finnigan Mat 251mass spectrometer. The reproducibility of the analytical procedurewas better than ±0.1‰ for calcite and ±0.2‰ for carbonate–Fe–Mg.All the samples were compared to a carbon dioxide reference obtainedfrom a calcite standard prepared at the same time. Thus, oxygen isotoperatios were recalculated taking into account the fractionation factor foracid decomposition at 50 °C (1.01057 for ankerite and dolomite) and at25 °C (1.01044 for calcite). C and O isotopemeasurements of carbonatematerials of the Gulf of Cadiz have been previously carried out with thismethod (Díaz-del-Río et al., 2003; González et al., 2009).

2.4. Organic compound analysis (lipids and amino acids)

To remove contaminants, we processed and cleaned the samplesin parallel with procedural blanks and we eliminated the externalsurface of the carbonate pipes. We placed the powdered samplesinto distilled water (Milli-Q) and left them for 2 h in an ultrasonicbath at room temperature. Then we concentrated and dried the mix-ture under vacuum. Subsequently, we powdered the samples at600 rpm using a planetary micro mill (Pulverisette 9, Fritsch, Idar-Oberstein, Germany).

Since our samples were extensively altered, to characterize thepreserved organic compounds we analyzed the samples by solidphase microextraction (SPME) coupled with gas chromatography–mass spectrometry (GC–MS), using the procedure of Menor-Salván

Table 1Sampling site data for the carbonate pipes studied in this work.

Dredgeno./cruise

Anastasyacruise

Volcanic orphysiographicunit

Geographiclocation

Depth (m) Number ofsamplesstudied

15 2001 Coruña mud-volcano 36°11′ N 814 17°32′ W

18 2001 Arcos mud-volcano 36°09′ N 880 27°33′ W

10 2000 Iberico mound 36°8′ N 870–950 47°43′ W

1, 2 2001 Cornide mound 36°7′ N 920–1145 67°37′ W

et al. (2008). GC–MS analyses were carried out on an AutosystemXL-Turbo Mass Gold (Perkin Elmer) with an Elite-5 column (cross-bond 5% diphenyl-95% dimethyl polysiloxane, 30 m×0.25 mmi.d.×0.25 μm film thickness) using He as carrier gas. The mass spec-trometer was operated with the following parameters: mode, EI+;ionization energy, 70 eV; m/z range, 30–600; transfer line tempera-ture, 300 °C. The temperature was programmed as follows: remain at40 °C for 4 min, increase from 40 to 150 °C at 15 °C/min, remain at150 °C for 2 min, increase from 150 to 255 °C at 5 °C/min, remain at255 °C for 15 min, increase from255 to 300 °C at 10 °C/min, and remainat 300 °C for 1 min.

In order to detect long-chain hydrocarbons and hopanes, we sub-jected the samples (20 g) to liquid–solid extraction with 100 ml of n-hexane:acetone (1:1) and three rounds of sonication. Combined ex-tracts (300 ml) were subjected to the following steps: (a) filtrationthrough a column with anhydrous sodium sulfate and freshly precipi-tated copper, (b) storage in hexane and concentration to 1 ml, (c) filtra-tion through a cleaned glass-fiber microfilter, and (d) concentration to25 μl under N2. We analyzed the extract (1 μl) by GC–MS as describedabove.

For the molecular characterization of analytes, we used the refer-ence spectra included in the NIST library and authenticated samples ac-quired from Sigma-Aldrich and Fluka. We considered an organiccompound to be positively determined only when the correlation be-tween the sample and reference spectra exceeded 85%.

We determined amino acid concentrations by reverse-phase high-pressure liquid chromatography (HPLC) according to Ruiz-Bermejo etal. (2007). Dissolved samples were hydrolyzed with 6 mol/l HCl at110 °C for 24 h in sealed vials and then freeze-dried to removewater and HCl. We identified amino acids by comparing their reten-tion times and UV absorption spectra with those of standards(Amino Acid Standards H, Pierce Chemical).

We used the molar percent of the amino acids to calculate the deg-radation index (DI), developed by Dauwe and Middelburg (1998) andDauwe et al. (1999). The DI assesses the diagenetic alteration of asample by comparing it to a set of 28 samples in different degradationstates and from different environments. We standardized the molarpercent of individual amino acids by subtracting the mean of allvalues from individual results and dividing by the standard deviationof all measurements. The DI integrates the amino acid data weightedby the factor coefficients of the first axis of the principal componentanalysis (PCA) of Dauwe et al. (1999) according to the formula:

DI ¼ ∑i

vari−AVG variSTD vari

� �·fac:coef :i

where vari is the original molar percent of each amino acid i, AVGvariand STDvari are the mean and standard deviations and fac.coef.i is thefactor coefficient of the first axis of the PCA of Dauwe et al. (1999).The DI indicates the cumulative deviation with respect to an assumedaverage molar composition, with negative values indicating moredegradation than the average and positive values indicating less.The DI varies from+1.5 for labile, fresh material to−3 for refractory,aged organic matter (Dauwe et al., 1999).

3. Results

3.1. Mineralogy, carbonate microfabrics and stable C and O isotopes

The pipes showed a wide variety of shapes and ranged in size fromseveral centimeters to a few decimeters. The external surface of thedry samples was brownish-gray and was densely perforated bysmall holes with diameters of 0.5–2 mm (Fig. 2A and B). Theseholes may be the result of chemical dissolution during exposure ofthe pipes to oxygenated waters. We observed grayish patches of sea-floor mud adhering to the walls and biological colonization by

Fig. 2. Photographs of cylindrical carbonate pipes from the Gulf of Cadiz. (A) and (B) Pipes with the external surface densely perforated by small holes. (C) Transverse slab showinginternal carbonate structure. (D) and (E) Pipes showing grayish patches of mud and biological colonization on their external walls.

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incrusting organisms (serpulid worm tubes and small corals) over theexternal walls of the pipes (Fig. 2D and E). Internal carbonate waslighter and less porous, containing quartz, feldspar and phyllosilicategrains, and well-preserved remains of foraminiferal and ostracodalshells (Figs. 2C and 3). As shown previously, iron sulfides and pseudo-morph oxyhydroxides were common components of the internalstructure of the pipes, forming framboidal agglomerates (Merineroet al., 2008, 2009).

XRD analyses revealed that the carbonate pipes consisted of Fe-rich dolomite, ankerite and Mg-rich calcite with an admixture of cal-cite, goethite and quartz. Concentrations of quartz and calcite werethe highest for pipes from the Arcos mud-volcano. Although carbon-ate pipes presented relatively homogeneous internal textures(Fig. 3), petrologic observations showed several internal carbonatefabrics: (1) small, elliptical pellets (5–20 μm) with a regular outlineand clear edges embedded in the microcrystalline carbonate matrix(Fig. 4A); (2) clots of irregular shape and size, cloudy internal texture

Fig. 3. Thin-section photomicrographs showing the internal texture of the carbonate pipes: a mand foraminiferal shells (F) filled with framboidal clusters of iron-oxyhydroxide pseudomorpchannel).

and indistinct margins, surrounded by microcrystalline carbonate(Fig. 4B); and (3) spheroidal nodules with rims of small framboidaliron minerals (diameterb2 μm) forming concentric layers and pre-senting a texture different from that of the surrounding carbonatematrix (Fig. 4C and D).

Carbon and oxygen isotopic analyses were performed on samplescollected from the Arcos mud-volcano and from the Iberico and Cor-nide mounds. The carbonate pipes were substantially depleted in13C: δ13C values varied from −9.24‰ to −38.36‰. Carbonate δ18Ovalues ranged from −0.99‰ to +6.65‰. Lower values of δ13C andδ18O were obtained for pipes from the Arcos mud-volcano, where cal-cite was present at higher concentrations.

3.2. Composition of lipids and amino acids

All samples showed similar amino acid compositions (Fig. 5). Ofthe 15 amino acids identified, the dominant amino acid in all samples

icrocrystalline carbonate matrix containing grains of quartz (Q), detrital iron-oxides (O),hs after pyrite (FR). Limits (L) are drawn between textures (pipe wall and filled internal

Fig. 4. Optical microscope images showing carbonate microfabrics of the studied pipes.Scale bar: 100 μm. (A) Peloids embedded in microcrystalline carbonate matrix.(B) Clotted microcrystalline carbonates. (C) A spherical nodule of microcrystalline car-bonate surrounded by framboidal iron minerals.

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was alanine, with a molar percent (mol%) varying from 36.5 to 44.9,followed by glycine (18.5–20.5 mol%), proline (3.3–10 mol%), histi-dine (2.3–6.4 mol%) and tyrosine (2.1–6.1 mol%). The remainingamino acids identified showed relative concentrations lower than5 mol%. DI values were similar for all samples, ranging from −0.43to −0.48.

The lipids identified included C10 to C24 n-alkanes without pre-dominance of odd-over-even carbon number, with a high abundanceof compounds with 17–20 carbons (Fig. 6). Classical liquid–solid ex-traction, together with the SPME technique, showed a lack of hydro-carbons with more than 27 carbons. Other compounds detected wereas follows: clusters of monomethyl-alkanes (MMA) and dimethyl-alkanes (DMA), phytanol (3,7,11,15-tetramethylhexadecanol), phytol(15-tetramethylhexadec-2-enol), and the regular isoprenoids farne-sane (2,6,10-trimethyldodecane), nor-pristane (2,6,10-trimethylpenta-decane), and pristane (2,6,10,14-tetramethylpentadecane). All samplescontained appreciable amounts of the head-to-tail isoprenoid phytane(2,6,10,14-tetramethylhexadecane), which partially co-eluted with aminor amount of the tail-to-tail irregular C20-isoprenoid crocetane(2,6,11,15-tetramethylhexadecane). Linear and methyl-branched mid-

chain fatty alcohols were found in all samples, from C10 to C18 n-alkanols.

4. Discussion

4.1. Relation between carbonate pipes and AOM

The observed depletion of 13C in the bulk carbonate is the first in-dication of the role played by the oxidation of methane in the forma-tion of the carbonate pipes studied: δ13C values varied from −9.24‰to −38.36‰. These δ13C values are similar to those of other carbon-ates from the Gulf of Cadiz (Díaz-del-Río et al., 2003; Stadnitskaia etal., 2008). Depletion of 13C in carbonates is widely accepted evidenceof a methane-related origin (Peckmann and Thiel, 2004). Methaneoxidation results in enhanced concentrations of isotopically depletedbicarbonate in pore waters, resulting in carbonate precipitation (Ritgeret al., 1987; von Rad et al., 1996; Peckmann and Goedert, 2005). Thesecarbonates inherit the stable isotope composition of their carbonsources (Campbell et al., 2002; Peckmann and Thiel, 2004). However,the 13C content results from mixing of different carbon sources duringcarbonate precipitation and the extent of mixing is hard to determine.Thus, an evaluation of fluid composition of δ13C carbonate valuesalone is problematic: carbon sources other than hydrocarbons are usu-ally relatively enriched in 13C and, thus, modern hydrocarbon-seepcarbonates generally show higher δ13C values than their hydrocarbonsource (Peckmann and Thiel, 2004). Therefore, if a certain amount ofmixing of different carbon pools is assumed, the lowest δ13C valuefrom the carbonate pipes (as low as−38.36‰) could indicate biogenicmethane as a carbon source. However, the overall δ13C values point tothermogenicmethane as themain carbon source according to previous-ly reported studies of methane origin in active mud-volcanoes of theGulf of Cadiz (Stadniskaia et al., 2006; Nuzzo et al., 2009).

The high δ18O value observed could be attributed to dolomite pre-cipitation from 18O-enriched diagenetic fluids, rather than to a tem-perature effect. Precipitation of calcite in equilibrium with marinebottom water (δ18O water≈0‰±0.2‰; Standard Mean OceanWater) at 4 °C has a δ18O value of approximately +3.2‰. Dolomiteshould be about 3‰ higher in δ18O than coexisting calcite, and Mg-calcite should have a δ18O value intermediate between these twophases at a given temperature (Anderson and Arthur, 1983; Land,1983). Calcite precipitated from fresh water or at elevated tempera-tures should have significantly lower isotopic values. Pore fluids de-rived from destabilization of clathrate or clay mineral dehydrationcould cause a small increase (1–2‰) in the δ18O of the local porefluids (Martin et al., 1996). Both processes occur in different areasof the Gulf of Cadiz (Somoza et al., 2003; León et al., 2006; Hensenet al., 2007) and could therefore be considered likely fluid sourcesfor precipitation of carbonate pipes. In addition, the lower values ofδ18O in samples from the Arcos mud-volcano may be explained bytheir higher content of calcite compared to other pipes in this study.

Precipitation of carbonates and iron sulfides is the main mineralog-ical consequence of AOM coupled with sulfate reduction at methane-seep sites (Peckmann and Thiel, 2004). Vasconcelos et al. (1995) andWarthmann et al. (2000) showed that, under anoxic conditions, bacte-rial sulfate reduction (BSR) overcomes the kinetic barrier to dolomiteformation by increasing the alkalinity of the carbonate and the sur-roundingmedium. As discussed above, the lower values of δ13C indicatethat a significant proportion of carbon in the bulk carbonate derivedfrom methane. At the same time, the previously reported abundanceof pyrite framboids in carbonate pipes of the Gulf of Cadiz (Merineroet al., 2008) indicates high local rates of sulfate reduction. Dolomite pre-cipitation is enhanced by the removal of seawater sulfate from pore-water, with concomitant increases in carbonate alkalinity (Baker andKastner, 1981). The most plausible conclusion from these findings isthat the dolomitic and ankeritic pipes in this study formed from porefluids in which the seawater sulfate had been locally consumed.

Fig. 5. Molar percents of the amino acids detected by HPLC in carbonate pipes from the Iberico and Cornide mounds and the Arcos mud-volcano. Abbreviations for amino acids areas follows: alanine (ALA), glycine (GLY), proline (PRO), histidine (HIS), tyrosine (TYR), aspartic acid (ASP), serine (SER), leucine (LEU), isoleucine (ILE), threonine, (THR), cysteine(CYS), glutamic acid (GLU), valine (VAL), phenylalanine (PHE), lysine (LYS).

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The observed carbonate microfabrics provide additional evidencefor the proposed relation between carbonate pipe formation andAOM. These features are frequently found in methane-related carbon-ates (e.g. Commeau et al., 1987; Hovland et al., 1987; Mazzini et al.,2004; Peckmann and Thiel, 2004; Han et al., 2008). Clotted microfab-rics and carbonate nodules are considered signs of microbial activity(Burne and Moore, 1987; Coleman, 1993; Raiswell and Fisher, 2000;

Fig. 6. Typical lipid distribution patterns in the carbonate pipes studied from the Gulf of Cacarbons of n-alkanes, Fa = farnesane, NP = nor-pristane, Pr = pristane, Ph = phytane,(m/z=184) showing phytanol, phytol and fatty alcohol peaks.

Peckmann et al., 2003), and their occurrence may be related tosmall variations in the chemical environment during carbonate pre-cipitation, caused by metabolic activity of methane-oxidizing organ-isms (Peckmann et al., 2002; Peckmann and Thiel, 2004; Buggischand Krumm, 2005). Pellets, for their part, could be interpreted as aproduct of bacterially induced precipitation of carbonates (Chavetz,1986).

diz. (A) GC–MS ion chromatogram (m/z=85) where numbers denote the number ofCr = crocetane, stars = unidentified methyl-alkanes. (B) GC–MS ion chromatogram

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4.2. Aerobic alteration of carbonate pipes

Before their collection in this study, the carbonate pipes sampledin the Gulf of Cadiz lay on the seafloor and were in contact with oxy-genated water (Díaz-del-Río et al., 2003), so aerobic alterationmay beexpected. Indeed, the pipes presented visible signs of bioerosion andalteration: the color and the small holes perforating the external surfacemay reflect dissolution due to the acidity generated by iron sulfideoxidation during the exposure of the pipes to oxygenated waters.Iron sulfide oxidation and pseudomorphic transformation into ironoxyhydroxides have been documented in previous mineralogicalstudies on these pipes (Merinero et al., 2008, 2009). Colonizationby incrusting organisms and grayish patches of mud attached tothe walls of the pipes are also signs of their residence on the seafloorand subsequent exposure to oxygenated waters.

This hypothesis of aerobic alteration of the pipes is supported byDI values calculated from the molar percent of the amino acids.Values ranged from−0.43 to−0.48, indicating degradation of the or-ganic matter preserved within the pipes, based on the DI interpreta-tion of Dauwe et al. (1999). Alanine was the most abundant aminoacid identified, followed by glycine. Both alanine and glycine are pref-erentially preserved in degraded organic matter, and their relativeconcentration increases with degradation (Lee and Cronin, 1984;Müller et al., 1986; Lee et al., 2000). On the other hand, valine, isoleu-cine, leucine and histidine were found at lowmol% in the pipes. Theseamino acids tend to be preferentially decomposed during organicmatter degradation (Hecky et al., 1973).

4.3. Sources of organic compounds

The inventory of organic substances preserved in methane-derivedcarbonates reflects the combination of allochthonous organic matterpreserved in carbonate porosity (e.g. filling foraminiferal tests), organiccompounds derived from methanotrophic and sulfate-reducing organ-isms and seeped hydrocarbons trapped within carbonates. In addition,biodegradation, thermal maturation, and aerobic oxidation influencethe nature of the organic substances preserved inside these carbonates,altering the composition and diluting their concentrations. These pro-cesses alter the preserved organic substances and their concentrationsto different extents over time and must be taken into account wheninterpreting the patterns observed in methane-derived carbonates.

Pristane, phytane, crocetane, farnesane and nor-pristane were theisoprenoids detected within carbonate pipes. Crocetane was the onlyspecific biomarker for AOM identified, and its precise contents are dif-ficult to establish since the abundance of phytane partially obscuredits presence. Studies of crocetane in fossil samples have shown thatthis compound can be used as a diagenetically stable fingerprint formethanotrophic archaea that thrive in methane-rich settings andthat aid in the formation of methane-derived carbonates (Peckmannand Thiel, 2004). The structure of crocetane, involving a tail-to-taillinkage of isoprene units, suggests an archaeal origin (Elvert et al.,1999, 2000; Peckmann et al., 1999; Thiel et al., 1999, 2001; Pancostet al., 2000). However, the association between crocetane and metha-notrophic archaea cannot be definitively demonstrated without deter-mining δ13C signatures. Pristane and phytane are typical diageneticproducts, respectively, of algal tocopherols and the phytol side-chainof chlorophyll (Didyk et al., 1978; Goossens et al., 1984). However,some authors, such as Birgel et al. (2006), have proposed that sedimen-tary phytane, pristane, nor-pristane and farnesane can be derived fromlipids of methane-oxidizing archaea. In particular, the most plausiblebiological precursors of phytane are archaeol and hydroxyarchaeolderivatives (Peckmann and Thiel, 2004). Similar scenarios may bevalid for pristane, nor-pristane and farnesane, which may derivefrom archaeal isopranyl lipids (Peckmann et al., 2002; Goedert etal., 2003). Therefore, we speculate that the observed pattern of iso-prenoids is derived from aerobic degradation of organic compounds

from sulfate dependent AOM with better preservation of archaealthan bacterial lipids. This is because the carbon skeletons of lipidsfrom SRB, due to their structure and low molecular weight, are lessresistant to biodegradation, thermal alteration, and weathering thanare isoprene-based archaeal biomarkers (Peters and Moldowan,1993). Consequently, these processes may erase evidence of anSRB source faster than evidence of archaeal chemofossils in methane-derived carbonates.

Other organic compounds detected inside carbonate pipes werephytanol, phytol and clusters of monomethyl-alkanes (MMA) and di-methyl-alkanes (DMA). Phytanol, a possible precursor of pristane(Peters et al., 2005), is a compound frequently found in AOM environ-ments of putative archaeal origin (Thiel et al., 1999; Hinrichs andBoetius, 2002; Peckmann et al., 2004). Oxic conditions promote theconversion of phytanol to pristane by oxidation of phytanol to phytanicacid, decarboxylation to pristene, and then reduction to pristane. There-fore, the detection of pristane in our organic analyses may be explainedby the exposure of the carbonate pipes to oxygenated waters andthe subsequent degradation of some precursor formed during AOMand initially trapped in the carbonate matrix. Detection of phytolcould be interpreted as allochthonous input because it is frequently de-rived from the ester-linked phytol moiety in chlorophyll and hence isassociated with photosynthetic activity of algae and higher plants. Thebranched hydrocarbons methylalkanes (MMAs) and dimethylalkanes(DMAs) are recognizable in ancient sediments. They are diageneticproducts derived from biosynthesized functionalized lipid precursors(Summons, 1987; Summons et al., 1988) and can be considered poten-tial biomarkers. Their use as specific, reliable biomarkers is limited byinadequate knowledge about the ranges inwhich they occur in modernsedimentary environments and by the likelihood that they serve as pre-cursors inmultiple biogenic pathways. Nevertheless, since they are pre-cursors ofmonomethyl fatty acids in some common SRB (Dowling et al.,1986),we speculate that a possible source of the short-chainMMAs andDMAs detected in our study is the degradation of long-chain fatty acidssynthesized by SRB initially present within the carbonate pipes.

Finally, we found linear and methyl branched mid-chain fatty alco-hols in all samples, from C10 to C18. Fatty alcohols frequently occur insediments at modern seeps and their presence has been attributed toSRB performing AOM (Orphan et al., 2001). However, they cannot beused to determine the specific groups of microorganisms involved inthe way that fatty acids can.

5. Summary and conclusions

Carbonate pipes sampled from the seafloor of the Gulf of Cadiz aredominated by authigenic microcrystalline Fe-rich dolomite, ankeriteand Mg-rich calcite. The δ13C values of carbonate pipes indicate for-mation from oxidation of methane. Their δ18O values agree with do-lomite precipitation, which may have occurred in a regime of sulfatedepletion compatible with BSR. The observed carbonate microfabricsof clotted carbonates and spherical nodules of microcrystalline car-bonate surrounded by framboidal iron minerals reflect the activityof microorganisms that drive AOM.

Despite the observed intensive aerobic alteration, some organiccompounds are preserved in these carbonate pipes. Crocetane wasthe only lipid identified from methanotrophic archaea. Although theobserved pattern of organic compounds (n-alkanes, regular isoprenoidsand alcohols) indicates aerobic alteration of the pipes, they are alsocompatible with an origin in AOM. Additional signs of exposure to oxy-genatedwaters and aerobic alteration are visible in the external surfaceof the pipes, such as small holes, grayish color, colonization and bioero-sion. Moreover, the degradation indexes calculated from themolar per-cent of amino acids preserved in pipes also indicate aerobic degradationof the trapped organic compounds.

Therefore, the conclusions of this study point to a significant mi-crobial contribution to the precipitation of the methane-derived

181R. Merinero et al. / Sedimentary Geology 263–264 (2012) 174–182

carbonate pipes sampled in the Gulf of Cadiz. We therefore speculatethat the precipitation is due, at least in part, to AOM coupled withBSR. These findings are not definitive, since aerobic alteration maskedthe lipid biomarkers of the specific microbes involved.

Acknowledgments

This study forms part of the work of the Research Group CAM-UCMand the work on terrestrial analogs for the exploration of Mars at theCentro de Astrobiología. The results were obtained within the frame-work of the European Science Foundation EuroCORE-EuroMARGINSprojects “MOUNDFORCE” (01-LEC-EMA06F, REN-2002-11668-E-MAR)and “MVSEIS” (01-LEC-EMA24F, REN-2002-11669-E-MAR). We thankall the scientific and technical personnel who participated in the ocean-ographic cruises Anastasya 2000 and 2001of theCornide de Saavedra re-search vessel. We are also very grateful to the “Centro de MicroscopíaElectrónica Luis Bru” (Complutense University of Madrid), to Drs. J.A.Martín-Rubí and F.J. González of the Geological Survey of Spain and toDr. A. Delgado of the “Estación Experimental del Zaidín” of the CSIC(Granada, Spain) for their contributions to the study. We wish tothank the collaborative efforts and insightful remarks of Dr. VictorDíaz del Río (Centro Oceanográfico de Málaga, Instituto Español deOceanografía). We appreciate the careful revision of Armando ChapinRodríguez who greatly aided in improving the final manuscript.

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