# Methane-derived stromatolitic carbonate crust from an active fluid seepage in the western basin of the Sea of Marmara: mineralogical, isotopic and molecular geochemical characterization

Akhoudas C. <fnref n="1" />, Chevalier N. <fnref n="1" />,*, Blanc-Valleron M-M <fnref n="2" />, Klein V. <fnref n="1" />, Mendez-Millan M. <fnref n="3" />, Demange J. <fnref n="1" />, Dalliah S. <fnref n="2" />, Rommevaux V. <fnref n="2" />, Boudouma O. <fnref n="2" />, Pierre C. <fnref n="1" />, Ruffine Livio <fnref n="4" />

## Abstract :

Cold seeps along the North Anatolian fault in the Sea of Marmara (Turkey) were explored during submersible dives of the Marsite cruise in November 2014 when sediments, pore waters and carbonate crusts were sampled at active fluid seeping sites. In this study, we investigate the mineralogy, carbon and oxygen isotopic compositions and the lipid biomarkers of a carbonate crust from the western Tekirdağ basin of the Sea of Marmara. This crust exhibits a laminated domal structure that resembles stromatolite. The mineralogy of authigenic seep-carbonate is mostly represented by aragonite associated with minor amounts of high-magnesian calcite. The abundance of pyrite associated with the authigenic seep-carbonate points to very intense bacterial sulfate reduction. The carbon (–42.6‰ to –34.4‰) and oxygen (–1.5‰ to +1.1‰) isotopic compositions of the authigenic seep-carbonate crust indicate that carbonate precipitation was related to anaerobic oxidation of methane and occurred in mixtures of bottom seawater with brackish water expelled from the underlying sediments. Abundant microbial lipid biomarkers with negative δ13C values (–121‰ to –96‰), confirm that anaerobic oxidation of methane (AOM) coupled with sulfate reduction, was mediated by methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB). Diagnostic lipid fingerprints indicate that ANME-2 archaea and associated SRB were the prevalent AOM-mediating consortia, which characterize moderate to high methane flow at this site. Moreover, changes in microbial lipid distribution within the carbonate crust suggest a variation in the intensity of methane emission.

**Keywords** : Marmara Sea, Tekirdağ Basin, methane, authigenic seep-carbonate, sulfate reduction, anaerobic oxidation of methane, stromatolitic structure, stable isotopes, lipid biomarkers

<footnote n="1">¹ Sorbonne Universités, UPMC, CNRS, IRD, MNHN, IPSL, LOCEAN, Paris, France</footnote>

<footnote n="2">² Sorbonne Universités, MNHN, UPMC, CNRS, CR2P, Paris, France</footnote>

<footnote n="3">³ Sorbonne Universités, IRD, UPMC, CNRS, MNHN, IPSL, LOCEAN, Bondy, France</footnote>

<footnote n="4">⁴ IFREMER, Département REM, Unité des Géosciences Marines, Plouzané, France</footnote>

<footnote>* Corresponding author : N. Chevalier, email address : nicolas.chevalier@upmc.fr</footnote>
Cold seeps are areas spread worldwide along continental margins where fluids rich in methane and hydrogen sulfide are present within the shallow sedimentary column and partly released into the water column (Suess, 2014). Most of the methane migrating upward within the sedimentary column is microbially consumed at the sulfate-methane transition zone (SMTZ) through anaerobic oxidation of methane (AOM), this process leading to the main methane sink on Earth (Hinrichs and Boetius, 2002; Knittel and Boetius, 2009; Reeburgh, 2007; Valentine and Reeburgh, 2000). It is commonly assumed that AOM involves microbial syntrophic consortia of methanotrophic archaea (ANME), and sulfate-reducing bacteria (SRB) (Boetius et al., 2000; Hinrichs et al., 1999; Orphan et al., 2002, 2001; Pancost et al., 2001, 2000). However, Milucka et al. (2012) have displayed that AOM is not necessarily associated with SBR. Molecular studies using fluorescence *in situ* hybridization and 16S ribosomal RNA gene sequence analysis revealed that AOM is mediated by at least three distinct clusters that are part of methanogenic archaea, ANME-1, ANME-2 and ANME-3 (Boetius et al., 2000; Hinrichs et al., 1999; Knittel et al., 2005; Niemann et al., 2006; Orphan et al., 2001). The production of bicarbonate ions during the AOM-SR process increases alkalinity in pore waters, which leads to the precipitation of authigenic carbonates (Claypool and Threlkeld 1983; Peckmann and Thiel, 2004). Indeed, many studies have shown that ¹³C-depleted lipid biomarkers synthesized by AOM microbial communities are contained in seep carbonates, supporting that these microbial consortia were involved in their precipitation (Aloisi et al., 2002, 2000; Bouloubassi et al., 2006; Chevalier et al., 2010, 2011; Gontharet et al., 2007; Greinert et al., 2002; Peckmann et al., 2001; Stadnitskaia et al., 2008). Moreover, the distribution of specific microbial lipid biomarkers (specific hydrocarbons, alcohols and fatty acids) and their carbon isotopic signature is frequently used to infer the taxonomic composition of microbial communities involved in AOM, which allow to distinguish the dominant AOM assemblages in cold seep ecosystems (see Blumenberg et al., 2004; Niemann and Elvert 2008 and references therein).

In the Sea of Marmara (SoM), a number of studies have shown that the authigenic carbonates in the cold seep environments were formed via the AOM process (Çağatay et al., 2011; Chevalier et al., 2011; Crémière et al., 2012). Moreover, the involvement of the dominant ANME-2 cluster associated with their specific SRB has been highlighted in several
cold seep-related carbonates, and also in sediments, using molecular and isotopic approaches (Chevalier et al., 2013, 2011).

In this study, we examine in detail a seafloor authigenic carbonate crust recovered at an active fluid venting site in the SoM during the “MarsiteCruise” expedition in 2014 onboard the R/V Pourquoi pas?. The main goal was to characterize the biogeochemical processes responsible for carbonate precipitation, using carbonate mineralogy, carbon and oxygen isotopic compositions of carbonate as well as the distribution and carbon isotopic compositions of lipid biomarkers.

# 1. Geological setting and sampling

The SoM is located between the Mediterranean Sea and the Black Sea in Turkish territory. It is composed of three distinct basins, named from west to east: Tekirdağ Basin, Central Basin and Çinarcik Basin (Fig. 1). The SoM is crossed along an east-west axis by the northern branch of the seismically active North Anatolian Fault (NAF) with the associated dense secondary-fault networks (Le Pichon et al., 2003, 2001). This whole fault system serves as conduits for fluid migration (Dupré et al., 2015). Bourry et al. (2009) and Ruffine et al. (this issue) reported that methane and other hydrocarbons from the seeping fluids in the Tekirdağ basin are mostly of thermogenic origin.

The studied carbonate crust was sampled during the dive MRS-DV4 with the remotely operated vehicle (ROV) Victor 6000 at an active seepage area located along the main fault scarp on the south-eastern part of the Tekirdağ Basin (40°48'18"N, 27°37'77"E; 1107 m water depth) (Fig. 1, 2A, 2B). The site was already visited in 2007 during the Marnaut cruise with the submersible Nautile, and referred to as “Jack the Smoker” (Armijo et al., 2005, Burnard et al., 2012), due to fluid emission from decimeter-high carbonate chimneys forming a mound-shaped construction. The carbonate crust, when sampled at the “Jack the Smoker” site, was partly buried in mud and partly overhanging the seafloor; its surface bathed with seawater was covered by orange biofilms whereas white bacterial mat covered the crust at the contact of the black reduced sediment (Çağatay et al., this issue; Fig. 2A and 2B). The distribution of crust elements dispersed on the seafloor suggests they were probably broken due to seismic activity along the faulted area described in Armijo et al. (2005).

Recent experiments with the deployment of an osmo-sampler over one year close to the “Jack the Smoker” vent have measured background flow rates of 5-20 cm/yr interrupted by episodic high flow rates (~1m/yr), (Tryon et al., 2012).
## 2. Analytical methods

### 2.1. Mineralogy and stable isotopes

Part of the frieze-dried carbonate crust was cut into several parallel slabs revealing their internal structure. One of these, showing the best textural details, was broken into 6 fragments (section 1', and 1 to 5) for analysis (Fig. 2C, Tables 1, 2 & 3). One or several small parts of each, was sampled for bulk mineralogical and carbon and isotopic analyses (Table 1). Microsamples were collected to analyse the carbon and oxygen isotopic variability on the nearby slab (Fig. 2C). The subsamples were ground in an agate mortar to obtain a fine powder. The total carbonate content of bulk sediment (in weight % of dry sediment, wt%) was measured by reacting 100 mg of the powder with 1 ml of HCl-8N in a manual calcimeter (precision of the measure: ± 1 %). The mineralogy of bulk sediment was determined through X-ray diffraction (XRD) of non-oriented powder, using a diffractometer (Bruker D2 Phaser) equipped with a Lynxeye detector, Cu-Kα radiation (λ=0.15406 Å) and Ni filter at 30 kV and 10 mA. The position of the major diffraction peak (d104) of calcite was used to estimate the relative mole% content of MgCO₃ in the crystal lattice (Goldsmith et al., 1961). When necessary, the main quartz peak was used as a reference to correct the position of the 104 peak of calcite. Mineral identification was made using the Diffrac.Eva v4.1 software and the ICDD PDF2 database. The relative percentages of aragonite and calcite within the subsamples were estimated by measuring the peak area with MacDiff software. Scanning electron microscope (SEM) observations and elemental analysis were made on a few selected zones to characterize the crystal morphology, microstructure and elemental composition of carbonate minerals and other authigenic phases.
The carbon and oxygen isotopic compositions of carbonates were measured on microsamples obtained by microdrill from a polished plate (Fig. 2C) as well as on the samples from the 6 sections (Table 1). The δ¹⁸O and δ¹³C values of these samples were obtained by reaction of 100 to 200 µg of powdered carbonate with anhydrous orthophosphoric acid for 12 minutes at 90°C. The released CO₂ was analyzed using an Isoprime Dual-Inlet isotopic ratio mass spectrometer equipped with a Multiprep system. The isotopic compositions are reported in delta (δ) units relative to the Vienna Pee Dee Belemnite reference (VPDB); sample bracketing was performed with the laboratory reference (Carrara marble; δ¹³C = +2.13‰ and δ¹⁸O = -1.83‰ VPDB).

## 2.2. Lipid biomarkers

Lipids were extracted from ≈ 5g (dried weight, dw) of powdered carbonate sub-samples corresponding to the 6 sections with a CH₂Cl₂/ MeOH mixture (2:1) by sonication (3 times). The total lipid extract was separated using a silica chromatography column into 3 fractions: F1 (hydrocarbons), F2 (ketones) and F3 (alcohols). An aliquot of the total extract was further saponified with 6% KOH in methanol (80°C, 1h) and extracted with hexane to yield the neutral lipids. Fatty acids were then obtained by acidification of the residual phase to pH = 1 and extracted with hexane. For GC analyses, fatty acids were methylated using Boron Trifluoride with Methanol (BF₃/MeOH) to form fatty acid methyl esters (FAMEs) and alcohols were derivatised (sylilation) with N,O-bis(tri-methylsilyl)trifluoracetamide (BSTFA) and pyridine to form trimethylsilyl-(TMS)ethers.

All fractions were analysed by gas chromatography (GC) on an Agilent 6890N instrument equipped with a column injector and a flame ionization detector (FID). A fused silica capillary column was used (30 m x 0.32 mm inner diameter, 0.25 µm film thickness) with helium as a carrier gas at 2.5 mL/min. The temperature program of the GC oven was as follows: (i) hydrocarbons: increase from 50 to 120°C at a rate of 30°C/min, then to 320°C at a rate of 5°C/min. The temperature was held at 320°C for 7 min, (ii) alcohols: increase from 50 to 100°C at a rate of 30°C/min, then to 150°C at a rate of 15°C/min followed by an increase to 300°C at a rate of 3°C/min. The temperature was then kept constant for 45 min. (iii) Fatty acids: increase from 50 to 100°C at a rate of 30°C/min, then to 310°C at 2°C/min, and finally maintained at 310°C for 35 min. Quantification was based on the GC-FID response relative
to internal standards (perdeuterated n-tetracosane, androstanol, heneicosanol and nonadecanoic acid) within the respective lipid fraction.

For structural identification of lipids, gas chromatography-mass spectrometry (GC-MS) analysis was performed using an Agilent 7890A GC equipped with a fused silica capillary column (30 m x 0.25 mm inner diameter, 0.25 µm film thickness) coupled to an Agilent 5975C MS instrument, which operated with an ionization energy of 70 eV with a scanning mass range of m/z 50-800. The oven temperature program was the same as for the GC analysis.

Compound-specific stable carbon isotope composition was measured using an online continuous flow gas chromatograph Trace-GC-Ultra coupled with an Isotopic Ratio Mass Spectrometer Delta V Plus via a combustion and conflow IV interface from Thermo Fischer Scientific (GC-C-IRMS). The GC was equipped with a 30 m fused silica capillary column (0.25 mm inner diameter, 0.25 µm film thickness). The temperature programs were the same as described above. Compositions are given in the delta notation as δ-value (‰), which is the relative deviation of the sample from the VPDB. The accuracy and reproducibility of the GC-C-IRMS measurements were assessed with a standard mixture of *n*-alkanes (C₁₆ to C₃₀) purchased from Schimmelmann (Indiana University) of known isotopic composition. The *n*-alkane standard mixture (type A6) was injected three times at the beginning of each run and then once every 3 samples. Analytical standard deviation over the entire analyses was of ± 0.6 ‰ (n = 60). Isotopic values of alcohols and fatty acids were corrected for the presence of carbon atoms added during silylation (δ¹³C BSTFA = -39.4 ± 0.1 ‰, n = 4) and methylation (δ¹³C BF₃-MeOH = -38.5 ± 0.2 ‰, n = 6).

## 3. Results

### 3.1. Macroscopic and microscopic observations

The rather flat top of the crust, just below the dark reduced sediment, has a rough surface showing circular to ovoid cavities partially connected (Fig. 3A), of infra to pluri-mm size, that might correspond to former voids (gas bubbles or bioturbation?) within the organic-rich sediment (Fig. 2C). A few cavities show an inner dark to orange coating. The resulting carbonate facies correspond to most of the grey-coloured part of the crust showing, in thin section, the coalescence of round 100 µm large micritic elements surrounded by fibrous aragonite, which forms a polygonal network of dark 10 µm particles (Fig. 3B & 3C). Pluri-
centimetric larger cavities, with a flat top, are also observed (former filled by the black reduced sediment), displaying pluri-mm to cm-sized dome-like structures on their borders.

Below the lower undulated surface of the larger cavities, domal structures develop downwards with alternating vacuolar layers, some of them parallel to their lower limit. Larger sequant pluri-mm large voids are also present. The more compact cream-coloured parts, especially at the bottom of the crust, exhibit thin laminations, made of 50 µm-thick orange or black layers separating irregular 200 µm to mm-thick layers (Fig. 3D). In thin section, the former ones look like biofilms whereas the thicker aragonite layers have a grass-like facies, composed of tubes of around 50 µm wide limited by darker spots corresponding to sulfides and/or organic matter.

During its growth, the crust maintained a contorted morphology prone to the living habitat of numerous gastropod and bivalve shells (mostly *Idas simpsoni*, S. Duperron, oral communication) that colonized the hard carbonate crust substrate and were later encrusted (Fig. 2C & 3A).

## 3.2. Mineralogy and carbon and oxygen isotope compositions of authigenic carbonate

The crust is mainly composed of carbonate (64 to 95 wt%) associated with few detrital minerals (mostly clay minerals), some authigenic pyrite, gypsum and sulfur. Aragonite (>77 %, Table 1) and high-magnesian calcite (with $d_{104}$ values of calcite ranging between 3.009 and 3.012 Å corresponding to ~8 mole% $MgCO_3$) are the dominant carbonate phases (Table 1).

The oxygen and carbon isotopic compositions of bulk carbonate from the sections vary from -0.2‰ to +1.1‰ and from -43.0‰ to -36.3‰, respectively (Table 1, Fig. 4). The microdrilled subsamples reveal a significant isotopic variability at the millimetric scale, with $\delta^{18}$O and $\delta^{13}$C values ranging from -1.5‰ to +1.0‰ and -41.6‰ to -37.6‰, respectively (Table 1, Fig. 4).

## 3.3. Lipid biomarkers

The lipid analysis reveals several archaeal compounds (Fig. 5A and 5B) including isoprenoidal dialkyl glycerol diethers represented by archaeol and *sn*-2-hydroxyarchaeol, saturated and unsaturated $C_{20}$ (crocetane), as well as polyunsaturated irregular $C_{25}$
isoprenoid alkenes with 2 to 4 unsaturations (2,6,10,15,19-pentamethylicosenes: PMI:2 to PMI:4) with PMI:3 being predominant. Heterogeneity in the biomarker concentration is observed from one carbonate section to another. Archaeol and *sn*-2-hydroxyarchaeol contents vary from 0.8 to 36.8 µg/g dw (Table 2). PMIs are the dominant compounds, except for sections 1 and 2 where archaeol is threefold more abundant. Another lipid with a phytanyl chain is also detected in lower amounts, i.e. phytanol (0.4 to 1.8 µg/g dw; Table 2).

All archaeal biomarkers in the crust are strongly depleted in ¹³C, suggesting incorporation of methane-derived carbon during their biosynthesis. PMIs are the most depleted compounds with δ¹³C values from -121‰ to -112‰ whereas phytanol displays slightly higher δ¹³C values (-98 to -96‰; Table 2). Crocetane/crocotene and isoprenoidal dialkyl glycerol diethers exhibit smaller variations with δ¹³C values from -116‰ to -110‰ and -106‰ to -101‰, respectively (Table 2). Moreover, apolar compounds (PMIs and their C₂₀ homologues) are more ¹³C-depleted than polar lipids (archaeol and *sn*-2-hydroxyarchaeol) with a δ¹³C offset of 10-15‰ (Table 2).

Various bacterial lipids are also present (Fig. 5B and 5C). Among them, the most abundant compounds are *i*-C₁₅:₀ and C₁₆:₁w₅ fatty acids (FAs) as well as a C₁₈-OH FA (Table 3). Terminally branched *ai*-C₁₅:₀ and *n*-C₁₄:₀ FAs occur at lower concentrations (3.5 to 33.4 µg/g dw; Table 3).

A sequence of *n*-alcohols (*n*-C₁₄:₀ to *n*-C₁₇:₁) with negative δ¹³C values (-90 to -70‰) are also detected in less abundance within the crust and they occur at concentrations ranging from 0.2 to 5.5 µg/g dw (Table 3). Other major bacterial biomarkers are dialkyl glycerol diethers (DAGEs; Chevalier et al., 2011 and references therein). The summed concentrations of the DAGEs are relatively high, ranging from 8.9 to 19.7 µg/g dw (Table 3).

All bacterial lipids show negative δ¹³C values with a range from -100‰ to -68‰. Moreover, *i*/*ai*-C₁₅:₀ and C₁₈-OH FAs show the most negative δ¹³C values around an average of -97‰ (Table 3). Compared to archaeal lipid biomarkers, bacterial lipids exhibit less negative δ¹³C values with δ¹³C offset of 10-40‰ between archaeal and bacterial lipids.

# 4. Discussion

## 4.1. Formation process of the authigenic seep-carbonate crust

The characteristic lithofacies of the studied seep-carbonate crust is the wavy laminae pattern similar to stromatolitic fabric (Fig. 2C, 3A, 3C, 3D). This stromatolitic morphology in seep-
carbonate, indicating a microbial activity, was first documented in marine authigenic seep-
carbonate at a depth of 4,850 m on the Aleutian accretionary margin (Greinert et al., 2002)
and more recently on offshore Nicaragua and Costa Rica in massive methane-derived
authigenic seep-carbonates from mound caps (Liebetrau et al., 2014). Typically, the
authigenic seep-carbonate crusts grow downwards in the sediment, and progressively make
hardgrounds at the seafloor, which prevent the upward fluid flow reaching the bottom water
except along conduits, fractures and where the seafloor remains uncemented.

Within the DV04-CC1 crust, authigenic seep-carbonates are mainly composed of aragonite (77 to 99 relative wt %) with minor to moderate contributions of high-Mg calcite (1 to 23 relative wt %). The mineralogy of carbonates is influenced by several physico-chemical conditions such as temperature, SO₄²⁻ concentrations and Mg²⁺/Ca²⁺ ratio (Burton and Walter, 1987; De Choudens-Sanchez and Gonzalez, 2009). The bottom water of the SoM is characterized by warm temperature (14.5°C) and SO₄²⁻ concentrations and Mg²⁺/Ca²⁺ ratio similar to those of Mediterranean bottom water (Ruffine et al., this issue; Tryon et al., 2010; Zitter et al., 2008) that would favour aragonite precipitation (Fig. 6A, 6B, 6D & 6H). High methane flow is also inferred to promote aragonite formation (Nöthen and Kasten, 2011).

The incorporation of dissolved inorganic carbon (DIC) produced by the oxidation of methane
is reflected in the carbon isotopic composition of the carbonate crust with δ¹³C values as low
as -42.6‰. The sub-samples of the carbonate crust also contain highly ¹³C-depleted
archaeal and bacterial lipids attesting for the incorporation of methane-derived carbon into
microbial biomass (e.g. Kellermann et al., 2012; Wegener et al., 2008). The presence of ¹³C-
depleted microbial lipid biomarkers thus indicate the involvement of methanotrophic archaea
(ANME) associated with sulfate reducers in the carbonate formation *via* anaerobic oxidation
of methane (AOM) coupled with bacterial sulfate reduction (SR). This is consistent with
previous studies reported for carbonate crust and sediment retrieved from cold seeps (Aloisi
et al., 2002; Bouloubassi et al., 2006; Chevalier et al., 2014, 2013, 2011, 2010; Elvert et al.,
2000, 1999; Gontharet et al., 2009; Greinert et al., 2002; Hinrichs et al., 2000, 1999; Pancost
and Sinninghe Damsté, 2003; Pancost et al., 2001, 2000; Stadnitskaia et al., 2008; Thiel et
al., 1999). The presence of pyrite (FeS₂) as framboids and single crystals (Fig. 6C, 6E, 6F,
6G, 6I) is also a signature of hydrogen sulfide formation associated with the AOM process
(Aloisi et al., 2000; Crémière et al., 2012; Greinert et al., 2002; Naehr et al., 2007; Pierre et
al., 2012). Indeed, pyrite is ubiquitous in anoxic sediments, where dissolved iron reacts with
hydrogen sulfide produced by bacterial sulfate reduction and is precipitated as sulfide
minerals (Berner, 1984). The abundance of pyrite in diagenetic carbonates from the Marmara
Sea confirms that microbial sulfate reduction was a very active process during carbonate
precipitation (Crémière et al., 2012). The presence of sulfur (Fig. 6E), iron oxides (Fig. 6G) and gypsum (Fig. 6E) in association with pyrite indicates that further oxidation of pyrite occurred when the carbonate crust was exposed to oxygenated bottom water conditions.

Although AOM/SR is the major process underlying the carbonate precipitation, methane-derived carbon may not be the only carbon source implied in the formation of the crust. Indeed, the difference between the carbon isotopic compositions of methane at this site (-66.1 < δ¹³C ‰ VPDB < -63.8; Ruffine et al., this issue) and those of the carbonate crust sections (Δδ¹³C = 21.2-28.8‰) indicate the admixture of at least one additional carbon source, especially from the pool of ¹³C-enriched DIC (e.g., Mazzini et al., 2004; Pierre and Fouquet, 2007). Such a DIC source would be issued from oxidation of non-methane hydrocarbons (Formolo et al., 2004), characterized by carbon isotopic composition between -37‰ and -29‰ (Ruffine et al, this issue). Moreover, the DIC produced by remineralization of organic matter from marine sediments (with an average δ¹³C value of -25‰) may be another carbon source (Crémière et al., 2012).

The oxygen isotopic composition of the bottom water of the Sea of Marmara (+1.6‰ VSMOW: Standard Mean Oceanic Water reference; Rank et al., 1999) was used to estimate the theoretical δ¹⁸O of aragonite that would precipitate in isotopic equilibrium at 14.5°C with the present-day bottom seawater, following the Böhm et al (2000) equation:

$$T^{\circ}\text{C} = 20 - 4.42 (\delta^{18}\text{O aragonite VPDB} - \delta^{18}\text{O water VSMOW})$$

Similar estimation was made for calcite, using the equation of Kim and O'Neil (1997), corrected by 0.06‰ for each mole % of MgCO₃ (Tarutani et al., 1969).

$$1000 \ln \alpha^{18}\text{O calcite-water} = 18.03 (10^3 T^{-1}) - 32.42$$

These equations give the δ¹⁸O values of +2.8‰ for aragonite and +2.0‰ for calcite containing 8% of MgCO₃, precipitated at equilibrium under the present-day bottom water conditions defined above.

In the studied carbonate crust, all samples exhibit δ¹⁸O values lower than these equilibrium values, thus indicating that carbonate precipitated with mixtures of bottom seawater with variable contribution of water with a negative δ¹⁸O value. The δ¹⁸O values of these mixed waters are estimated to range between -0.1‰ and -2.7‰, for aragonite with δ¹⁸O values ranging between +1.1‰ and -1.5‰ precipitated at the present-day bottom temperature. Indeed, the sampling site, “Jack the Smoker”, is characterized by expulsion of brackish fluids issued from underlying sediments deposited during the last glacial period when brackish
conditions existed in the Sea of Marmara (Çağatay et al., this issue; Crémière et al., 2012; Tryon et al., 2010; Zitter et al., 2008). These brackish waters are characterized by low Cl⁻ content (231 mM) and negative δ¹⁸O values (-5.5‰ VSMOW), as measured in pore waters of a core located on the Western High (Aloisi et al., 2015). This allows for the estimation of the fraction of the brackish water that mixed with bottom seawater to form the mixture from which the studied carbonate crust precipitated; the fraction is estimated between 0.3 and 0.6.

## 4.2. Composition of AOM microbial communities during stromatolitic carbonate precipitation

Specific lipid biomarker fingerprints obtained from the six subsamples clearly indicate the presence of both archaeal clusters, ANME-1 and ANME-2. Generally, cold seep settings host several ANME groups, knowing that only one dominates the microbial community. Each assemblage produces specific ¹³C-depleted lipids in different amounts, which determine the dominant ANME group involved in AOM (Niemann and Elvert, 2008). PMIs are synthesized by both ANME-1 and ANME-2 clades (Blumenberg et al., 2004). However, the δ¹³C offset of 10-15‰ observed between PMIs and isoprenoidal glycerol diethers, point to a preferential synthesis of PMIs by ANME-2 archaeal lineage (Blumenberg et al., 2005; Chevalier et al., 2014; Niemann and Elvert, 2008). This result is supported by the abundance of crocetane and its unsaturated counterparts together with PMIs. The dominance of ANME-2 community is also supported in subsamples 1' and 4 by the prevalence of sn-2-hydroxyarchaeol over archaeol (ratios 3 and 1.22, respectively; Niemann and Elvert, 2008). On the contrary, archaeol predominates over sn-2-hydroxyarchaeol in subsamples 1, 2, 3 and 5 (ratios 0.11, 0.02, 0.42 and 0.66 respectively), which suggests a higher proportion of ANME-1 archaea in the microbial pool in these subsamples (Blumenberg et al., 2004; Niemann and Elvert, 2008).

The bacterial patterns are in line with the ANME-2 inferred archaeal lipid data. The low ratio of *ai*-C₁₅:₀ relative to *i*-C₁₅:₀ FAs (0.3 to 0.6) and the presence of compounds such as *n*-C₁₄:₀ and C₁₆:₁w₅ were observed in the samples. These lipid patterns suggest a dominance of SRB eco-types that have been found associated with ANME-2 (Hinrichs et al., 2000; Niemann and Elvert, 2008), in accordance with the distribution of non-isoprenoid dialkyl glycerol ethers (DAGEs) with negative δ¹³C values in carbonate crusts from the three basins of the Marmara Sea (Chevalier et al., 2011). In this study, DAGEs were attributed to sulfate reducing bacteria in syntrophy with ANME-2 archaea.
The presence of ¹³C-depleted phytanol, a methanotroph archaea-derived lipid (Ge et al., 2015; Niemann and Elvert, 2008; Thiel et al., 1999) is also observed. However, the δ¹³C values (-98 < δ¹³C ‰ VPDB < -96) of phytanol are slightly heavier than in other archaeal biomarkers. This isotopic composition might be induced by a small contribution of phytanol synthetized by microorganisms different to ANMEs (Elvert et al., 2000). The distribution of short-chain *n*-alcohols present in the studied carbonate crust (from *n*-C₁₄:₀ to *n*-C₁₇:₁) showed δ¹³C values ranging between -70‰ and -85‰. These compounds have more positive δ¹³C values than archaeal lipids, which indicate different source organisms and thus, a potential origin from sulfate reducing bacteria with an autotrophic growth on methane-derived dissolved inorganic carbon (e.g. Hinrichs and Boetius, 2002; Wegener et al., 2008 and references therein). Another peculiar result is the presence of a C₁₈ FAME with a hydroxyl group (C₁₈-OH FA) in high quantities throughout the carbonate and characterized by very negative δ¹³C values. The specific isotopic signature strongly indicates the incorporation of methane-derived carbon by their microbial precursor. The presence of this fatty acid was never previously observed in AOM-related environments (sediment core or carbonate). Their δ¹³C values identical to those of archaeol and *sn*-2-hydroxyarchaeol, combined with the absence of any isoprenoidal carbon structure, discards an archaeal origin (Volkman, 2006). Indeed, long-chain hydroxyl fatty acids can be related to methanotrophic bacteria (Skerratt et al., 1992). Despite the fact that there is no available DNA data to confirm this precursor, it may be suggested that methanotrophic bacteria could be a potential biological source of the ¹³C-depleted C₁₈-OH FA.

Previous studies have shown that the taxonomy of microbial consortia in AOM environments
depends on specific conditions such as temperature, methane concentration and degree of
anoxia (Knittel et al., 2005; Nauhaus et al., 2002, 2005; Treude et al., 2003). For instance,
the intensity of methane flow would be a factor to explain the presence of different types of
ANME (ANME-1 vs. ANME-2) (Blumenberg et al., 2004; Rossel et al., 2011; Stadnitskaia et
al., 2008). The lipid biomarker patterns of the studied carbonate crust indicate a
predominance of ANME-2 consortia suggesting that the carbonate crust was formed via
AOM during moderate to high methane flux conditions (Blumenberg et al., 2004; Knittel et al.,
2005; Rossel et al., 2011; Stadnitskaia et al., 2008). However, we observe changes in the
distribution of lipid biomarkers with a net dominance of ANME-2 community associated with
their specific SRB in sections 1' and 4, and higher proportions of ANME-1/SRB in the other
sections. These differences in ¹³C-depleted microbial lipid composition might indicate the
variability of *in situ* methane and sulfate concentration during these different precipitation
phases, probably due to fault dynamics (Ruffine et al., 2015). This model was also
demonstrated in the study of Stadnitskaia et al. (2008) with a clear change of archaeal
community during the formation of an AOM-related carbonate crust that was attributed to a
change in methane flow within the sediments. While changes of methane flux and anoxic
conditions may control the AOM community structure, the δ¹³C values of archaeal and
bacterial lipid biomarkers remain here relatively constant during the carbonate precipitation
phases, which indicates a common source of methane used by AOM-related microbial
assemblages during carbonate formation.

Consequently, the distribution of microbial lipid biomarkers could be used as a molecular
proxy to estimate the paleochanges of methane emission and hypoxic/anoxic conditions in
relation with seismic dynamics.

Conclusions

Observations of an authigenic carbonate crust taken from an active methane seep in the
Tekirdağ Basin of the Sea of Marmara, together with mineralogical, isotopic and molecular
geochemical analyses, made it possible to highlight the biogeochemical processes
responsible for its formation. The major conclusions of this study are:

(i) Microbial activity is indicated by a laminated structure similar to a stromatolitic fabric.

(ii) A high amount of ¹³C-depleted archaeal and bacterial lipids associated with very negative δ¹³C values of CaCO₃ in the different sections, and microprofiles, provide evidence for a carbonate formation by way of AOM and SR activity, mediated by ANME methanotrophic archaea and SRB members.

(iii) Lipid biomarker patterns indicate that the dominant AOM assemblages consist of ANME-2 archaea and associated SRB, suggesting carbonate formation in environments with moderate to high methane flow. Moreover, a variability of microbial community composition is observed through the carbonate structure, confirming changes in methane fluxes emitted along the NAF.

(iv) The presence of a ¹³C-depleted hydroxyl fatty acid (C₁₈-OH) in this cold seep carbonate probably indicate that bacteria are involved during anaerobic methane oxidation.
Acknowledgements

We thank the captain and his crew on-board the RV Pourquoi pas? and the team of the ROV Victor-6000 crew for their technical support and advice. Financial support was provided by the European programme "MARsite", under the call ENV.2012.6.4-2: "Long-term monitoring experiment in geologically active regions of Europe prone to natural hazards: the Supersite concept". We also thank the ALYSES facility (IRD-UPMC) that was supported by grants from Région Ile-de-France for molecular isotope analyses. Thanks are also due to Alison Chalm who kindly helped to improve the English text. Finally, we thank Tobias Himmler, two other anonymous reviewers and the guest editor for their constructive comments that have greatly contributed in improving the manuscript.

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## Figure captions

Figure 1. Bathymetric map of the Sea of Marmara showing the tectonic framework and the carbonate crust location (MRS-DV04-CC1). NAF: North Anatolian Fault.

Figure 2. Seafloor pictures from ROV dive MRS-DV595-04. A: general view of Jack the smoker site (white chimney at arrow), B: close-up view of carbonate crust with indication of location of sample MRS-DV04-CC1 (arrow). C: Cross-cut section of the crust MRS-DV04-CC1 with the position of the studied subsamples.

Figure 3. Thin section (3.5 x 5 cm) of the carbonate crust MRS-DV04-CC1. A: scanned view showing stromatolitic fine laminae molding the lower irregular surface of the crust with two shells of *Idas*, vacuolar structure of most of the internal part of the crust. B: microscopic view in polarized light showing spheroidal aggregates of aragonite and overgrowths of palissadic aragonitic cement. C: microscopic view in polarized light of bottom of the crust showing spherolitic, micritic, and botryoidal facies of aragonite. D: microscopic view in natural light showing micritic grass-like facies of aragonite and late botryoidal acicular aragonite.

Figure 4. Cross-plot of δ¹⁸O-δ¹³C values of carbonate subsamples of crust MRS-DV04-CC1. The filled circles correspond to the subsamples from sections; the open circles correspond to the microdrilled subsamples (see Table1). The dashed lines represent the d18O values of aragonite and high-Mg calcite precipitated in isotopic equilibrium with the present-day bottom seawater.

Figure 5. Reconstructed gas chromatograms of the hydrocarbon (A), alcohol (B) and fatty acid (C) fractions extracted from the crust MRS-DV04-CC1 (Tekirdag Basin in the SoM).
Fatty acids and alcohols were analyzed as their methyl ester and TMS derivatives, respectively. Black circles indicate *n*-alkanes, straight-chain alcohols and saturated fatty acids. IS: internal standards.

Figure 6. SEM photographs (back-scattered imagery) of the crust MRS-DV04-CC1. A & B: micritic tubular and granular facies of the cream-colored aragonitic layers (longitudinal & transversal cross sections). C: pyrite-rich layer with isolated crystals and framboids, gastropod mold and foraminifer remains. D: aragonite needles radiating around circular microcrystalline core (10-50 µm of diameter) of spheroidal aggregates. E: pyrite (bright), sulfur (light grey), and secondary gypsum (medium grey) crystals. F: secondary gypsum crystals and pyrite. G: more or less oxidized pyrite associated with aragonite needles. H: radiating aragonite needles. I: sub-rhomboedral crystals of high-magnesian calcite and aragonite needles, pyrite framboids, nearby biofilm.

<table><caption>Table 1. Mineralogy and isotopic composition of the carbonate crust MRS-DV04-CC1 subsamples.</caption><thead><tr><th>MRS-DV04-01<br>subsamples</th><th>Total<br>carbon<br>te wt%</th><th>Aragoni<br>te rel<br>wt%</th><th>Mg-<br>Calcit<br>e rel<br>wt%</th><th>d<sub>104</sub><br>Mg-<br>Calcit<br>e</th><th>MgC<br>O<sub>3</sub><br>mole<br>%</th><th>Detrital minerals</th><th>Pyrit<br>e</th><th>Accessor<br>y<br>minerals</th><th>δ<sup>18</sup>O<br>‰<br>VPD<br>B</th><th>δ<sup>13</sup>C<br>‰<br>VPD<br>B</th></tr></thead><tbody><tr><td>zone A: dark slightly<br>cemented</td><td>64</td><td>99</td><td>1</td><td>3,000</td><td>12</td><td>Clay (tr), Quartz?</td><td>++</td><td>Gypsum<br>(tr)</td><td>1,1</td><td>-43,0</td></tr><tr><td>zone B: grey</td><td>85</td><td>89</td><td>11</td><td>3,012</td><td>8</td><td>Clay (tr), Quartz?</td><td>tr</td><td></td><td>0,5</td><td>-39,5</td></tr><tr><td>zone C: cream-colored</td><td>93</td><td>96</td><td>4</td><td>3,009</td><td>9</td><td>Clay (tr), Quartz?</td><td>tr</td><td></td><td>0,2</td><td>-36,3</td></tr><tr><td>zone 1, light grey</td><td>94</td><td>92</td><td>8</td><td>3,011</td><td>8</td><td>Clay (tr), Quartz?</td><td>+</td><td></td><td>0,8</td><td>-41,9</td></tr><tr><td>zone 1, darker grey</td><td>88</td><td>88</td><td>12</td><td>3,011</td><td>8</td><td>Clay (tr), Fd (?),<br>Quartz?</td><td>++</td><td></td><td>0,6</td><td>-42,6</td></tr><tr><td>zone 2, grey and white</td><td>91</td><td>92</td><td>8</td><td>3,011</td><td>8</td><td>Quartz (tr), Clay (?)</td><td>+</td><td></td><td>0,5</td><td>-39,3</td></tr><tr><td>zone 3, light grey</td><td>93</td><td>85</td><td>15</td><td>3,011</td><td>8</td><td>Clay (tr), Quartz (tr)</td><td>+</td><td></td><td>0,2</td><td>-38,2</td></tr><tr><td>zone 3, darker grey</td><td>93</td><td>77</td><td>23</td><td>3,011</td><td>8</td><td>Clay (tr), Quartz (tr)</td><td>+</td><td></td><td>0,3</td><td>-39,4</td></tr><tr><td>zone 3, lower laminated<br>part with orange biofilm</td><td>90</td><td>92</td><td>8</td><td>3,011</td><td>8</td><td>Quartz (tr), Clay (?)</td><td>+</td><td>Sulfur (tr)</td><td>0,7</td><td>-37,7</td></tr><tr><td>zone 3, grey and white</td><td>94</td><td>92</td><td>8</td><td>3,011</td><td>8</td><td>Quartz (tr), Clay (?)</td><td>+</td><td></td><td>0,6</td><td>-40,5</td></tr><tr><td>zone 4, grey</td><td>94</td><td>89</td><td>11</td><td>3,011</td><td>8</td><td>Clay?, Quartz?</td><td>+</td><td></td><td>-0,3</td><td>-41,0</td></tr><tr><td>zone 4, dark grey granular</td><td>94</td><td>91</td><td>9</td><td>3,012</td><td>8</td><td>Clay?, Quartz?, Fd?</td><td>+</td><td></td><td>0,5</td><td>-39,6</td></tr><tr><td>zone 5, border of the crust</td><td>88</td><td>94</td><td>6</td><td>3,010</td><td>8</td><td>Clay?, Quartz?</td><td>+</td><td>Sulfur (tr)</td><td>0,6</td><td>-38,6</td></tr></tbody></table>
<table><caption>Table 2. Concentrations and carbon isotopic compositions of archaeal lipids extracted from the carbonate crust MRS-DV04-CC1 subsamples.</caption><caption>with orange biofilm</caption><tbody><tr><td>zone 5, cream-colored</td><td>91</td><td>95</td><td>5</td><td>3,010</td><td>8</td><td>Clay?, Quartz?</td><td>+</td><td>0,3</td><td>-37,3</td></tr><tr><td>zone 5, grey</td><td>95</td><td>85</td><td>15</td><td>3,011</td><td>8</td><td>Quartz (tr), Clay (?)</td><td>+</td><td>-0,2</td><td>-40,6</td></tr><tr><td>micro-sample (1)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0,8</td><td>-41,6</td></tr><tr><td>micro-sample (2)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0,4</td><td>-38,8</td></tr><tr><td>micro-sample (3)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-1,5</td><td>-39,3</td></tr><tr><td>micro-sample (4)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0,0</td><td>-40,1</td></tr><tr><td>micro-sample (5)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0,1</td><td>-37,6</td></tr><tr><td>micro-sample (6)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-0,1</td><td>-38,1</td></tr><tr><td>micro-sample (7)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0,5</td><td>-38,8</td></tr><tr><td>micro-sample (8)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0,6</td><td>-40,3</td></tr><tr><td>micro-sample (9)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1,0</td><td>-41,3</td></tr><tr><td>micro-sample (10)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0,2</td><td>-40,2</td></tr><tr><td>micro-sample (11)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0,9</td><td>-39,1</td></tr></tbody></table>

<table><thead><tr><th rowspan="2"></th><th colspan="2">section 1</th><th colspan="2">section 1'</th><th colspan="2">section 2</th><th colspan="2">section 3</th><th colspan="2">section 4</th><th colspan="2">section 5</th></tr><tr><th>µg/g<br>dw</th><th>δ<sup>13</sup>C<br>(‰)</th><th>µg/g<br>dw</th><th>δ<sup>13</sup>C<br>(‰)</th><th>µg/g<br>dw</th><th>δ<sup>13</sup>C<br>(‰)</th><th>µg/g<br>dw</th><th>δ<sup>13</sup>C<br>(‰)</th><th>µg/g<br>dw</th><th>δ<sup>13</sup>C<br>(‰)</th><th>µg/g<br>dw</th><th>δ<sup>13</sup>C<br>(‰)</th></tr></thead><tbody><tr><td>Archaeol</td><td>23.8</td><td>-106</td><td>7.5</td><td>-106</td><td>36.8</td><td>-105</td><td>4.9</td><td>-106</td><td>8.6</td><td>-104</td><td>6.2</td><td>-105</td></tr><tr><td>Sn-2-<br>hydroxyarchaeol</td><td>2.6</td><td>-102</td><td>22.4</td><td>-101</td><td>0.8</td><td>nd</td><td>2.1</td><td>-101</td><td>10.4</td><td>-100</td><td>4.1</td><td>-101</td></tr><tr><td>Phytanol</td><td>0.8</td><td>-98</td><td>0.9</td><td>-97</td><td>1.8</td><td>-97</td><td>0.4</td><td>-96</td><td>0.8</td><td>-96</td><td>0.6</td><td>-96</td></tr><tr><td>Crocetane (Cr:0)</td><td>2.9</td><td>-116</td><td>3.6</td><td>-113</td><td>5.0</td><td>-115</td><td>2.8</td><td>-115</td><td>3.9</td><td>-114</td><td>5.2</td><td>-115</td></tr><tr><td>Cr:1</td><td>0.6</td><td>-113</td><td>0.7</td><td>-110</td><td>1.3</td><td>-114</td><td>0.7</td><td>-113</td><td>1.0</td><td>-113</td><td>1.7</td><td>-114</td></tr><tr><td>Cr:2</td><td>0.2</td><td>nd</td><td>0.1</td><td>nd</td><td>0.4</td><td>nd</td><td>0.4</td><td>nd</td><td>0.5</td><td>nd</td><td>1.1</td><td>nd</td></tr><tr><td>PMI:2</td><td>0.9</td><td>nd</td><td>2.1</td><td>nd</td><td>0.9</td><td>-112</td><td>1.3</td><td>-113</td><td>1.4</td><td>-113</td><td>1.9</td><td>nd</td></tr><tr><td>PMI:3</td><td>4.4</td><td>-120</td><td>14.3</td><td>-116</td><td>8.7</td><td>-118</td><td>9.0</td><td>-117</td><td>9.3</td><td>-117</td><td>10.6</td><td>-117</td></tr><tr><td>PMI:4</td><td>2.6</td><td>-121</td><td>9.0</td><td>-116</td><td>4.9</td><td>-121</td><td>5.6</td><td>-116</td><td>5.5</td><td>-116</td><td>6.8</td><td>-116</td></tr><tr><td>ΣPMIs</td><td>7.9</td><td></td><td>25.4</td><td></td><td>14.5</td><td></td><td>15.9</td><td></td><td>16.2</td><td></td><td>19.3</td><td></td></tr></tbody></table>

nd: not
determined

<table><caption>Table 3. Concentrations and carbon isotopic compositions of bacterial lipids extracted from the carbonate crust MRS-DV04-CC1 subsamples.</caption><thead><tr><th rowspan="2"></th><th colspan="2">section 1</th><th colspan="2">section 1'</th><th colspan="2">section 2</th><th colspan="2">section 3</th><th colspan="2">section 4</th><th colspan="2">section 5</th></tr><tr><th>µg/g dw</th><th>δ<sup>13</sup>C (‰)</th><th>µg/g dw</th><th>δ<sup>13</sup>C (‰)</th><th>µg/g dw</th><th>δ<sup>13</sup>C (‰)</th><th>µg/g dw</th><th>δ<sup>13</sup>C (‰)</th><th>µg/g dw</th><th>δ<sup>13</sup>C (‰)</th><th>µg/g dw</th><th>δ<sup>13</sup>C (‰)</th></tr></thead><tbody><tr><td colspan="14"><strong>FAMES</strong></td></tr><tr><td>n-C<sub>14:0</sub></td><td>3.5</td><td>-68</td><td>13.7</td><td>-74</td><td>6.6</td><td>-76</td><td>4.5</td><td>-72</td><td>6.8</td><td>nd</td><td>8.0</td><td>-70</td></tr><tr><td>i-C<sub>15:0</sub></td><td>15.9</td><td>-100</td><td>58.3</td><td>-99</td><td>38.6</td><td>-100</td><td>21.3</td><td>-99</td><td>38.3</td><td>-99</td><td>36.3</td><td>-97</td></tr><tr><td>ai-C<sub>15:0</sub></td><td>6.6</td><td>-89</td><td>33.4</td><td>-97</td><td>16.0</td><td>-96</td><td>8.3</td><td>-95</td><td>12.7</td><td>-93</td><td>15.5</td><td>-94</td></tr><tr><td>n-C<sub>16:1ω5</sub></td><td>23.9</td><td>-89</td><td>91.6</td><td>-86</td><td>45.1</td><td>-89</td><td>29.8</td><td>-87</td><td>39.4</td><td>-87</td><td>55.8</td><td>-87</td></tr><tr><td>C<sub>18-OH</sub></td><td>21.6</td><td>-98</td><td>159.3</td><td>-97</td><td>67.2</td><td>-100</td><td>26.9</td><td>-98</td><td>35.6</td><td>-97</td><td>69.7</td><td>-97</td></tr><tr><td colspan="14"><strong>Alcohols</strong></td></tr><tr><td>n-C<sub>14:0</sub></td><td>0.9</td><td>-76</td><td>0.9</td><td>-76</td><td>1.4</td><td>-75</td><td>0.7</td><td>-74</td><td>0.6</td><td>-70</td><td>0.6</td><td>-71</td></tr><tr><td>n-C<sub>15:0</sub></td><td>0.5</td><td>-79</td><td>0.4</td><td>-79</td><td>0.5</td><td>-78</td><td>0.3</td><td>-77</td><td>0.2</td><td>nd</td><td>0.3</td><td>-81</td></tr><tr><td>n-C<sub>16:1</sub></td><td>3.6</td><td>-80</td><td>4.3</td><td>-80</td><td>5.5</td><td>-80</td><td>3.0</td><td>-80</td><td>2.2</td><td>-79</td><td>2.7</td><td>-80</td></tr><tr><td>n-C<sub>16:0</sub></td><td>3.4</td><td>-77</td><td>3.0</td><td>-77</td><td>4.6</td><td>-77</td><td>2.3</td><td>-76</td><td>1.9</td><td>-72</td><td>2.4</td><td>-74</td></tr><tr><td>n-C<sub>17:1</sub></td><td>0.5</td><td>-85</td><td>0.4</td><td>-83</td><td>0.7</td><td>-81</td><td>0.3</td><td>-81</td><td>0.3</td><td>nd</td><td>0.6</td><td>-83</td></tr><tr><td>DAGEs (1 and 2)</td><td>8.9</td><td>-90</td><td>19.7</td><td>-89</td><td>13.6</td><td>-87</td><td>10.1</td><td>-87</td><td>10.5</td><td>-86</td><td>15.6</td><td>-88</td></tr></tbody></table>

nd: not determined
<figure><img src="image_1.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_2.png" /><figcaption>**Figure 2**</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 4</figcaption></figure>

Akhoudas et al.
<figure><img src="image_5.png" /><figcaption>Figure 5</figcaption></figure>

Akhoudas et al.
<figure><img src="image_6.png" /><figcaption>Figure 6</figcaption></figure>